Primers and probes for detection and discrimination of types and subtypes of influenza viruses

ABSTRACT

Methods of detecting influenza, including differentiating between type and subtype are disclosed, for example to detect, type, and/or subtype an influenza infection. A sample suspected of containing a nucleic acid of an influenza virus, is screened for the presence or absence of that nucleic acid. The presence of the influenza virus nucleic acid indicates the presence of influenza virus. Determining whether the influenza virus nucleic acid is present in the sample can be accomplished by detecting hybridization between an influenza specific probe, influenza type specific probe, and/or subtype specific probe and an influenza nucleic acid. Probes and primers for the detection, typing and/or subtyping of influenza virus are also disclosed. Kits and arrays that contain the disclosed probes and/or primers also are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/182,852, filed Jun. 15, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/056,810, filed Oct. 17, 2013, now U.S. Pat. No.9,382,592, which is a divisional of U.S. patent application Ser. No.13/554,782, filed Jul. 20, 2012, now U.S. Pat. No. 8,568,981, which is adivisional of U.S. patent application Ser. No. 12/191,186, filed Aug.13, 2008, now U.S. Pat. No. 8,241,853, which is a continuationapplication of International Application No. PCT/US2007/003646, filedFeb. 12, 2007, published under PCT Article 21(2) in English, and claimsthe benefit of U.S. Provisional Application No. 60/772,806, filed Feb.13, 2006, all of which applications are incorporated by reference hereinin their entirety.

FIELD

This disclosure relates to primers and probes for detecting one or moretypes or subtypes of influenza virus, as well as kits including theprobes and primers and methods of using the probes and primers.

BACKGROUND

Influenza virus types A and B are members of the orthomyxoviridae familyof viruses that cause influenza infection. The infective potential ofinfluenza is frequently underestimated and can result in high morbidityand mortality rates, especially in elderly persons and in high-riskpatients, such as the very young and immuno-compromised. Influenza A andB viruses primarily infect the nasopharyngeal and oropharyngeal cavitiesand produce highly contagious, acute respiratory disease that results insignificant morbidity and economic costs. Typical influenza viralinfections in humans have a relatively short incubation period of 1 to 2days, with symptoms that last about a week including an abrupt onset offever, sore throat, cough, headache, myalgia, and malaise. When asubject is infected with a highly virulent strain of influenza thesesymptoms can progress rapidly to pneumonia and in some circumstancesdeath. Pandemic outbreaks of highly virulent influenza present a seriousrisk to human and animal health worldwide.

The immunodominant antigens present on the surface of influenza virusesare hemagglutinin (HA or H) and neuraminidase (N). Genetic reassortmentbetween human and avian influenza viruses can result in a virus with anovel hemagglutinin of avian origin, against which humans lack immunity.In the 20^(th) century, the pandemics of 1918, 1957, and 1968 were theresult of such antigenic shifts. The avian influenza outbreaks of theearly 21_(st) century caused by H5N1, H7N7, and H9N2 subtype influenzaviruses, and their infection of humans have created a new awareness ofthe pandemic potential of influenza viruses that circulate in domesticpoultry. The economic impact of a major influenza pandemic has beenestimated to be up to $165 billion in the United States alone, with asmany as 200,000 deaths, 730,000 hospitalizations, 42 million outpatientvisits, and 50 million additional illnesses.

To combat influenza infection, neuraminidase inhibitors have recentlybeen developed. Clinical studies carried out for the Food and DrugAdministration's (FDA) approval of neuraminidase inhibitors in theUnited States showed that successful treatment primarily depends onprompt treatment after the first clinical symptoms occur. Unfortunately,it is generally not possible for even experienced medical professionalsto reliably diagnose influenza solely on the basis of the patient'sclinical symptoms because other viruses which infect the nasal orpharyngeal cavity, such as adenoviruses, parainfluenza viruses, orrespiratory syncitial viruses (RS viruses), cause similar symptoms. Toeffectively treat influenza infection it is necessary to begin treatmentas early as possible in the development of the infection, ideally uponthe onset of non-virally specific clinical symptoms.

A variety of methods have been used to detect influenza virusesclinically. In one example, influenza viruses are detected by culturingsamples obtained from a subject on mammalian cells such as Madine-DarbyCanine Kidney cells (MDCK). Culturing mammalian cells is costly and timeconsuming (taking up to 14 days) and is thus not of immediate relevancefor the diagnosis of the individual patient. Other methods of detectionthat have been developed include immunofluorescence assays (IFA), enzymeimmunoassays (EIA), and enzyme-linked immunosorbent assays (ELISA) thatuse antibodies specific to influenza virus antigens. Culture andserological tests require long completion times (5 days to 2 weeks) withpotentially greater exposure of technical personnel to infectiousagents. Immunoassays are generally faster (30 minutes to 4 hours) butoften require substantial sample handling and rely on subjectivedetermination of results by technical personnel. Furthermore, thesetests typically are not capable of rapidly differentiating between theinfluenza types and subtypes, some of which have pandemic potential.

Hence the need remains for a test that provides sensitive, specificdetection of influenza virus types and subtypes in a relatively shorttime, so that diagnosis is completed in sufficient time to permiteffective treatment of an infected person.

SUMMARY

The present disclosure relates to methods of detecting the presence ofan influenza virus in a sample, such as a biological sample obtainedfrom a subject. The disclosed methods can be used for diagnosing aninfluenza infection in a subject suspected of having an influenzainfection by analyzing a biological specimen from a subject to detect abroad variety of influenza types and subtypes. Alternatively, the methodcan be used to quickly identify particular types and subtypes ofinfluenza virus, particularly viruses that may be involved in pandemics.In addition, panels of probes are provided that permit the rapidevaluation of a subject with an apparent viral illness by quicklydetermining whether the illness is caused by a virulent pandemic virus(such as an H5 virus, for example H5N1). This rapid evaluation involvesruling out the presence of the pandemic virus (for example by positivelyidentifying a non-pandemic pathogen such as influenza type B), ruling inthe presence of the pandemic virus (for example by identifying apandemic viral pathogen such as an H5 virus, for example H5N1), or acombination of both.

In some embodiments, the method involves hybridizing an influenzanucleic acid to an influenza specific probe between 20 and 40nucleotides in length, and detecting hybridization between influenzanucleic acid and the probe. In some embodiments, the probe is detectablylabeled. In some embodiments, the probe is capable of hybridizing underconditions of very high stringency to an influenza nucleic acid sequenceset forth as SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. Inspecific embodiments, the probe includes a nucleic acid sequence that isat least 95% identical to a nucleic acid sequence set forth as SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ IDNO:29, SEQ ID NO:32, SEQ ID NO:35, or SEQ ID NO:38.

The present disclosure also relates to methods of detecting and/ordiscriminating between influenza viral types and/or subtypes. Thesemethods include contacting a sample with a probe that is specific for aninfluenza type and/or subtype and detecting the hybridization betweenthe influenza type and/or subtype specific probe. Detection ofhybridization between an influenza type and/or subtype specific probeand an influenza nucleic acid indicates that the influenza type and/orsubtype is present in the sample. In some embodiments, the methodsinclude detecting an influenza viral type and/or subtype. In oneexample, detecting hybridization to a nucleic acid sequence at least 95%identical to SEQ ID NO:8 indicates the presence of influenza type A. Inanother example, detecting hybridization to a nucleic acid sequence atleast 95% identical to SEQ ID NO:11 indicates the presence of influenzasubtype H1. In another example, detecting hybridization to a nucleicacid sequence at least 95% identical to SEQ ID NO:14 indicates thepresence of influenza subtype H3. In another example, detectinghybridization to a nucleic acid sequence at least 95% identical to SEQID NO:19 indicates the presence of influenza subtype H5. In anotherexample, detecting hybridization to a nucleic acid sequence at least 95%identical to SEQ ID NO:24 indicates the presence of influenza subtypeH5. In another example, detecting hybridization to a nucleic acidsequence at least 95% identical to SEQ ID NO:29 indicates the presenceof influenza type B. In another example, detecting hybridization to anucleic acid sequence at least 95% identical to SEQ ID NO:32 indicatesthe presence of influenza subtype North American H7. In another example,detecting hybridization to a nucleic acid sequence at least 95%identical to SEQ ID NO:35 indicates the presence of influenza subtypeEuropean H7. In yet another example, detecting hybridization to anucleic acid sequence at least 95% identical to SEQ ID NO:38 indicatesthe presence of subtype Asian H9 in the sample.

In some embodiments, the methods disclosed herein include amplifying theinfluenza nucleic acids with at least one primer specific for aninfluenza nucleic acid. In some embodiments, the primer specific for aninfluenza nucleic acid is 15 to 40 nucleotides in length and is capableof hybridizing under very high stringency conditions to an influenzavirus nucleic acid sequence set forth as SEQ ID NO:42, SEQ ID NO:43, SEQID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, or SEQ ID NO:50. In some embodiments, the primer specific for aninfluenza nucleic acid is 15 to 40 nucleotides in length and includes anucleic acid sequence at least 95% identical to the nucleotide sequenceset forth as SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 22, SEQ IDNO:23, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:37.

In some embodiments, the influenza nucleic acid is amplified using atleast one primer, such as a pair of primers, specific for an influenzatype and/or subtype. In some examples, a primer specific for influenzatype A includes a nucleic acid sequence at least 95% identical to thenucleic acid sequence set forth as one of SEQ ID NO:3 or SEQ ID NO:4. Inother examples, a primer specific for influenza subtype H1 includes anucleic acid sequence at least 95% identical to the nucleic acidsequence set forth as one of SEQ ID NO:9 or SEQ ID NO:10. In otherexamples, a primer specific for influenza subtype H3 includes a nucleicacid sequence at least 95% identical to the nucleic acid sequence setforth as one of SEQ ID NO:12 or SEQ ID NO:13. In other examples, aprimer specific for influenza subtype H5 includes a nucleic acidsequence at least 95% identical to the nucleic acid sequence set forthas one of SEQ ID NO:17 or SEQ ID NO:18. In other examples, a primerspecific for influenza subtype H5 includes a nucleic acid sequence atleast 95% identical to the nucleic acid sequence set forth as one of SEQID NO:22 or SEQ ID NO:23. In other examples, a primer specific forinfluenza type B includes a nucleic acid sequence at least 95% identicalto the nucleic acid sequence set forth as one of SEQ ID NO:26 or SEQ IDNO:28. In other examples, a primer specific for influenza subtype NorthAmerican H7 includes a nucleic acid sequence at least 95% identical tothe nucleic acid sequence set forth as one of SEQ ID NO:30 or SEQ IDNO:31. In other examples, a primer specific for influenza subtypeEuropean H7 includes a nucleic acid sequence at least 95% identical tothe nucleic acid sequence set forth as one of SEQ ID NO:33 or SEQ IDNO:34. In other examples, a primer specific for influenza subtype AsianH9 includes a nucleic acid sequence at least 95% identical to thenucleic acid sequence set forth as one of SEQ ID NO:36 or SEQ ID NO:37.

Additional methods for detecting, typing, and/or subtyping an influenzavirus in a sample include hybridizing nucleic acids in the sample to atleast one influenza type and/or subtype specific probe arrayed in apredetermined array with an addressable location.

This disclosure also relates to probes capable of hybridizing to anddiscriminating between influenza nucleic acids from specific typesand/or subtypes. In some embodiments, these probes are between 20 and 40nucleotides in length and capable of hybridizing under very highstringency conditions to an influenza nucleic acid sequence set forth asSEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In severalexamples, these probes are between 20 and 40 nucleotides in length andinclude a nucleic acid sequence set forth as SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:32,SEQ ID NO:35, or SEQ ID NO:38.

This disclosure also relates to primers capable of hybridizing to andamplifying an influenza nucleic acid, such as an influenza nucleic acidspecific to an influenza type and/or subtype. In some embodiments, theseprimers are between 20 and 40 nucleotides in length and capable ofhybridizing under very high stringency conditions to an influenzanucleic acid sequence set forth as SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, or SEQ ID NO:50. In several examples, these primers are 15 to 40nucleotides in length and include a nucleic acid sequence at least 95%identical to a nucleic acid sequence set forth as SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:17, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:36, or SEQ ID NO:37.

The disclosure also provides devices, such as arrays, as well as kitsfor detecting, typing, and/or subtyping an influenza virus in a samplesuspected of containing an influenza virus.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a generalized procedure forhybridizing an influenza specific probe to an influenza nucleic acid.

FIG. 2 is a schematic representation of a generalized procedure forhybridizing an influenza specific probe to an influenza nucleic acid,wherein the influenza nucleic acid is initially a double strandednucleic acid.

FIG. 3 is a schematic representation of a generalized procedure forhybridizing and detecting influenza using an influenza specific TAQMAN®probe.

FIG. 4 is a graph of theoretical data generated from real-time reversetranscriptase polymerase chain reaction (rt RT-PCR) using TAQMAN®probes.

FIG. 5A is a graph of a dilution series of SYBER green binding toinfluenza nucleic acids amplified with influenza A specific primers.

FIG. 5B is a graph of the dissociation curves obtained from the metingof influenza nucleic acids amplified with influenza A specific primersas shown in FIG. 5A.

FIG. 5C is a plot of the Ct values extracted from the graphs shown inFIG. 5A, as a function of concentration of template nucleic acidconcentration.

FIG. 6A is a graph of data obtained from rt RT-PCRs run on a dilutionseries of influenza nucleic acids using an influenza A specificprobe/primer set.

FIG. 6B is a plot of the Ct values obtained from the graphs shown inFIG. 6A, as a function of template nucleic acid concentration.

FIG. 7 is a graph of the data obtained from a series of rt RT-PCRs runat annealing temperatures ranging from 50-62.5° C.

FIG. 8A is a graph of data generated from rt RT-PCRs of a sampleobtained from a subject using the indicated influenza type and subtypeTAQMAN® probes.

FIG. 8B is a graph of data generated from rt RT-PCRs of a sampleobtained from a subject using the indicated influenza type and subtypeTAQMAN® probes.

FIG. 8C is a graph of data generated from rt RT-PCRs of a sampleobtained from a subject using the indicated influenza type and subtypeTAQMAN® probes.

FIGS. 9A-9F show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza type A M gene (SEQ ID NO: 42) used to design the disclosedinfluenza type A specific probes and primers.

FIGS. 10A-10I show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza type B NS gene (SEQ ID NO: 43) used to design the disclosedinfluenza type B specific probes and primers.

FIGS. 11A-11F show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza subtype H1 HA gene (SEQ ID NO: 44) used to design thedisclosed influenza subtype H1 specific probes and primers.

FIGS. 12A-12F show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza subtype H3 HA gene (SEQ ID NO: 45) used to design thedisclosed influenza subtype H3 specific probes and primers.

FIGS. 13A-13I show a table showing the consensus sequence and variationspresent in the specified influenza isolates for a region of theinfluenza subtype H5 HA gene (SEQ ID NO: 46) used to design thedisclosed influenza subtype H5 specific probes and primers that.

FIGS. 14A-14L show a table showing the consensus sequence and variationspresent in the specified influenza isolates for a region of theinfluenza subtype H5 HA gene (SEQ ID NO: 47) used to design thedisclosed influenza subtype H5 specific probes and primers.

FIGS. 15A-15B show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza subtype North American H7 HA gene (SEQ ID NO: 48) used todesign the disclosed influenza subtype North American H7 specific probesand primers.

FIGS. 16A-16C show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza subtype European H7 HA gene (SEQ ID NO: 49) used to design thedisclosed influenza subtype European H7 specific probes and primers.

FIGS. 17A-17I show a table showing the consensus sequence and variationspresent in the specified influenza isolates for the region of theinfluenza subtype Asian H9 HA gene (SEQ ID NO: 50) used to design thedisclosed influenza subtype Asian H9 specific probes and primers.

DETAILED DESCRIPTION I. Explanation of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “a probe” includes single or pluralprobes and can be considered equivalent to the phrase “at least oneprobe.”

As used herein, the term “comprises” means “includes.” Thus, “comprisinga probe” means “including a probe” without excluding other elements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the invention, thefollowing explanations of terms are provided:

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and veterinary subjects, such as birds.

Amplification: To increase the number of copies of a nucleic acidmolecule. The resulting amplification products are called “amplicons.”Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Other examples of in vitro amplification techniques include quantitativereal-time PCR; reverse transcriptase PCR; real-time reversetranscriptase PCR (rt RT-PCR); nested PCR; strand displacementamplification (see U.S. Pat. No. 5,744,311); transcription-freeisothermal amplification (see U.S. Pat. No. 6,033,881, repair chainreaction amplification (see WO 90/01069); ligase chain reactionamplification (see EP-A-320 308); gap filling ligase chain reactionamplification (see U.S. Pat. No. 5,427,930); coupled ligase detectionand PCR (see U.S. Pat. No. 6,027,889); and NASBA™ RNA transcription-freeamplification (see U.S. Pat. No. 6,025,134) amongst others.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA alsocan contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA can besynthesized in the laboratory by reverse transcription from RNA.

Change: To become different in some way, for example to be altered, suchas increased or decreased. A detectable change is one that can bedetected, such as a change in the intensity, frequency or presence of anelectromagnetic signal, such as fluorescence. In some examples, thedetectable change is a reduction in fluorescence intensity. In someexamples, the detectable change is an increase in fluorescenceintensity.

Complementary: A double-stranded DNA or RNA strand consists of twocomplementary strands of base pairs. Complementary binding occurs whenthe base of one nucleic acid molecule forms a hydrogen bond to the baseof another nucleic acid molecule.

Normally, the base adenine (A) is complementary to thymidine (T) anduracil (U), while cytosine (C) is complementary to guanine (G). Forexample, the sequence 5′-ATCG-3′ of one ssDNA molecule can bond to3′-TAGC-5′ of another ssDNA to form a dsDNA. In this example, thesequence 5′-ATCG-3′ is the reverse complement of 3′-TAGC-5′.

Nucleic acid molecules can be complementary to each other even withoutcomplete hydrogen-bonding of all bases of each molecule. For example,hybridization with a complementary nucleic acid sequence can occur underconditions of differing stringency in which a complement will bind atsome but not all nucleotide positions.

Detect: To determine if an agent (such as a signal or particularnucleotide or amino acid) is present or absent. In some examples, thiscan further include quantification. For example, use of the disclosedprobes in particular examples permits detection of a fluorophore, forexample detection of a signal from an acceptor fluorophore, which can beused to determine if a nucleic acid corresponding to nucleic acid of aninfluenza virus is present.

Electromagnetic radiation: A series of electromagnetic waves that arepropagated by simultaneous periodic variations of electric and magneticfield intensity, and that includes radio waves, infrared, visible light,ultraviolet light, X-rays and gamma rays. In particular examples,electromagnetic radiation is emitted by a laser, which can possessproperties of monochromaticity, directionality, coherence, polarization,and intensity. Lasers are capable of emitting light at a particularwavelength (or across a relatively narrow range of wavelengths), forexample such that energy from the laser can excite a donor but not anacceptor fluorophore.

Emission or emission signal: The light of a particular wavelengthgenerated from a fluorophore after the fluorophore absorbs light at itsexcitation wavelengths.

Excitation or excitation signal: The light of a particular wavelengthnecessary to excite a fluorophore to a state such that the fluorophorewill emit a different (such as a longer) wavelength of light.

Fluorophore: A chemical compound, which when excited by exposure to aparticular stimulus such as a defined wavelength of light, emits light(fluoresces), for example at a different wavelength (such as a longerwavelength of light).

Fluorophores are part of the larger class of luminescent compounds.Luminescent compounds include chemiluminescent molecules, which do notrequire a particular wavelength of light to luminesce, but rather use achemical source of energy. Therefore, the use of chemiluminescentmolecules (such as aequorin) eliminates the need for an external sourceof electromagnetic radiation, such as a laser.

Examples of particular fluorophores that can be used in the probesdisclosed herein are known to those of skill in the art and includethose provided in U.S. Pat. No. 5,866,366 to Nazarenko et al., such as4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), QFITC (XRITC), -6-carboxy-fluorescein(HEX), and TET (Tetramethyl fluorescein); fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosanilin; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate,N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); sulforhodamine B;sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine101 (Texas Red); riboflavin; rosolic acid and terbium chelatederivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein;boron dipyrromethene difluoride (BODIPY); acridine; stilbene;6-carboxy-X-rhodamine (ROX); Texas Red; Cy3; Cy5, VIC® (AppliedBiosystems); LC Red 640; LC Red 705; and Yakima yellow amongst others.

Other suitable fluorophores include those known to those skilled in theart, for example those available from Molecular Probes (Eugene, Oreg.).In particular examples, a fluorophore is used as a donor fluorophore oras an acceptor fluorophore.

“Acceptor fluorophores” are fluorophores which absorb energy from adonor fluorophore, for example in the range of about 400 to 900 nm (suchas in the range of about 500 to 800 nm). Acceptor fluorophores generallyabsorb light at a wavelength which is usually at least 10 nm higher(such as at least 20 nm higher) than the maximum absorbance wavelengthof the donor fluorophore, and have a fluorescence emission maximum at awavelength ranging from about 400 to 900 nm. Acceptor fluorophores havean excitation spectrum which overlaps with the emission of the donorfluorophore, such that energy emitted by the donor can excite theacceptor. Ideally, an acceptor fluorophore is capable of being attachedto a nucleic acid molecule.

In a particular example, an acceptor fluorophore is a dark quencher,such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACKHOLE QUENCHERS™ (Glen Research), ECLIPSE™ Dark Quencher (EpochBiosciences), or IOWA BLACK™ (Integrated DNA Technologies). A quenchercan reduce or quench the emission of a donor fluorophore. In such anexample, instead of detecting an increase in emission signal from theacceptor fluorophore when in sufficient proximity to the donorfluorophore (or detecting a decrease in emission signal from theacceptor fluorophore when a significant distance from the donorfluorophore), an increase in the emission signal from the donorfluorophore can be detected when the quencher is a significant distancefrom the donor fluorophore (or a decrease in emission signal from thedonor fluorophore when in sufficient proximity to the quencher acceptorfluorophore).

“Donor Fluorophores” are fluorophores or luminescent molecules capableof transferring energy to an acceptor fluorophore, thereby generating adetectable fluorescent signal from the acceptor. Donor fluorophores aregenerally compounds that absorb in the range of about 300 to 900 nm, forexample about 350 to 800 nm. Donor fluorophores have a strong molarabsorbance coefficient at the desired excitation wavelength, for examplegreater than about 10³ M⁻¹ cm⁻¹.

Fluorescence Resonance Energy Transfer (FRET): A spectroscopic processby which energy is passed between an initially excited donor to anacceptor molecule separated by 10-100 Å. The donor molecules typicallyemit at shorter wavelengths that overlap with the absorption of theacceptor molecule. The efficiency of energy transfer is proportional tothe inverse sixth power of the distance (R) between the donor andacceptor (1/R⁶) fluorophores and occurs without emission of a photon. Inapplications using FRET, the donor and acceptor dyes are different, inwhich case FRET can be detected either by the appearance of sensitizedfluorescence of the acceptor or by quenching of donor fluorescence. Forexample, if the donor's fluorescence is quenched it indicates the donorand acceptor molecules are within the Förster radius (the distance whereFRET has 50% efficiency, about 20-60 Å), whereas if the donor fluorescesat its characteristic wavelength, it denotes that the distance betweenthe donor and acceptor molecules has increased beyond the Försterradius, such as when a TAQMAN® probe is degraded by Taq polymerasefollowing hybridization of the probe to a target nucleic acid sequenceor when a hairpin probe is hybridized to a target nucleic acid sequence.In another example, energy is transferred via FRET between two differentfluorophores such that the acceptor molecule can emit light at itscharacteristic wavelength, which is always longer than the emissionwavelength of the donor molecule.

Examples of oligonucleotides using FRET that can be used to detectamplicons include linear oligoprobes, such as HybProbes, 5′ nucleaseoligoprobes, such as TAQMAN® probes, hairpin oligoprobes, such asmolecular beacons, scorpion primers and UniPrimers, minor groove bindingprobes, and self-fluorescing amplicons, such as sunrise primers.

Hybridization: The ability of complementary single-stranded DNA or RNAto form a duplex molecule (also referred to as a hybridization complex).Nucleic acid hybridization techniques can be used to form hybridizationcomplexes between a probe or primer and a nucleic acid, such as aninfluenza nucleic acid. For example, a probe or primer (such as any ofSEQ ID NOS:3-38) having some homology to an influenza nucleic acidmolecule will form a hybridization complex with an influenza nucleicacid molecule (such as any of SEQ ID NOS:42-50). Hybridization occursbetween a single stranded probe and a single stranded target nucleicacid (such as an influenza nucleic acid), as illustrated in FIG. 1. Whenthe target nucleic acid is initially one strand of a duplex nucleic acidthe duplex must be melted (at least partially) for the probe tohybridize. This situation is illustrated in FIG. 2.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength (suchas the Na+ concentration) of the hybridization buffer will determine thestringency of hybridization. Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning, second edition, ColdSpring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). Thefollowing is an exemplary set of hybridization conditions and is notlimiting:

Very High Stringency (Detects Sequences that Share at Least 90%Identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share at Least 80% Identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share at Least 50% Identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

The probes and primers disclosed herein can hybridize to influenzanucleic acids under low stringency, high stringency, and very highstringency conditions.

Influenza Virus: Influenza viruses are enveloped negative-sense virusesbelonging to the orthomyxoviridae family. Influenza viruses areclassified on the basis of their core proteins into three distincttypes: A, B, and C. Within these broad classifications, subtypes arefurther divided based on the characterization of two antigenic surfaceproteins hemagglutinin (HA or H) and neuraminidase (NA or N). While Band C type influenza viruses are largely restricted to humans, influenzaA viruses are pathogens of a wide variety of species including humans,non-human mammals, and birds. Periodically, non-human strains,particularly of avian influenza, have infected human populations, insome cases causing severe disease with high mortality. Recombinationbetween such avian strains and human strains in coinfected individualshas given rise to recombinant influenza viruses to which immunity islacking in the human population, resulting in influenza pandemics. Threesuch pandemics occurred during the twentieth century (pandemics of 1918,1957, and 1968) and resulted in numerous deaths worldwide.

Influenza viruses have a segmented single-stranded (negative orantisense) genome. The influenza virion consists of an internalribonucleoprotein core containing the single-stranded RNA genome and anouter lipoprotein envelope lined by a matrix protein. The segmentedgenome of influenza consists of eight linear RNA molecules that encodeten polypeptides. Two of the polypeptides, HA and NA include the primaryantigenic determinants or epitopes required for a protective immuneresponse against influenza. Based on the antigenic characteristics ofthe HA and NA proteins, influenza strains are classified into subtypes.For example, recent outbreaks of avian influenza in Asia have beencategorized as H5N1, H7N7, and H9N2 based on their HA and NA phenotypes.

HA is a surface glycoprotein which projects from the lipoproteinenvelope and mediates attachment to and entry into cells. The HA proteinis approximately 566 amino acids in length, and is encoded by anapproximately 1780 base polynucleotide sequence of segment 4 of thegenome. Polynucleotide and amino acid sequences of HA (and otherinfluenza antigens) isolated from recent, as well as historic, avianinfluenza strains can be found, for example in the GENBANK® database(available on the world wide web at ncbi nlm nih.gov/entrez) or theInfluenza Sequence Database of Los Alamos National Laboratories (LANL)(available on the world wide web at http://www.flu.lanl.gov). Forexample, recent avian H1 subtype HA sequences include: AY038014, andJ02144; recent avian H3 subtype HA sequences include: AY531037, M29257,and U97740; H5 subtype HA sequences include: AY075033, AY075030,AY818135, AF046097, AF046096, and AF046088; recent H7 subtype HAsequences include: AJ704813, AJ704812, and Z47199; and, recent avian H9subtype HA sequences include: AY862606, AY743216, and AY664675.

In addition to the HA antigen, which is the predominant target ofneutralizing antibodies against influenza, the neuraminidase (NA)envelope glycoprotein is also a target of the protective immune responseagainst influenza. NA is an approximately 450 amino acid protein encodedby an approximately 1410 nucleotide sequence of influenza genome segment6. Recent pathogenic avian strains of influenza have belonged to the N1,N7 and N2 subtypes. Exemplary NA polynucleotide and amino acid sequencesinclude for example, N1: AY651442, AY651447, and AY651483; N7: AY340077,AY340078 and AY340079; and, N2: AY664713, AF508892, and AF508588.

The remaining segments of the influenza genome encode the internalproteins. PB2 is a 759 amino acid polypeptide which is one of the threeproteins which comprise the RNA-dependent RNA polymerase complex. PB2 isencoded by approximately 2340 nucleotides of the influenza genomesegment 1. The remaining two polymerase proteins, PB1, a 757 amino acidpolypeptide, and PA, a 716 amino acid polypeptide, are encoded by a 2341nucleotide sequence and a 2233 nucleotide sequence (segments 2 and 3),respectively.

Segment 5 consists of about 1565 nucleotides encoding an about 498 aminoacid nucleoprotein (NP) protein that forms the nucleocapsid. Segment 7consists of an about 1027 nucleotide sequence of the M gene, whichencodes the two matrix proteins; an about 252 amino acid M1 protein, andan about 96 amino acid M2 protein, which is translated from a splicedvariant of the M RNA. Segment 8 consists of the NS gene, which encodestwo different non-structural proteins, NS1 and NS2.

Isolated: An “isolated” biological component (such as a nucleic acid)has been substantially separated or purified away from other biologicalcomponents in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA, RNA, and proteins. Nucleic acidsthat have been “isolated” include nucleic acids purified by standardpurification methods. The term also embraces nucleic acids prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids, such as probes and primers. Isolated does not requireabsolute purity, and can include nucleic acid molecules that are atleast 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% oreven 100% isolated.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy. For example, a label can be attached to anucleotide, thereby permitting detection of the nucleotide, such asdetection of the nucleic acid molecule of which the nucleotide is apart. Examples of labels include, but are not limited to, radioactiveisotopes, enzyme substrates, co-factors, ligands, chemiluminescentagents, fluorophores, haptens, enzymes, and combinations thereof.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed for example in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989)and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1998).

Nucleic acid (molecule or sequence): A deoxyribonucleotide orribonucleotide polymer including without limitation, cDNA, mRNA, genomicDNA, and synthetic (such as chemically synthesized) DNA or RNA. Thenucleic acid can be double stranded (ds) or single stranded (ss). Wheresingle stranded, the nucleic acid can be the sense strand or theantisense strand. Nucleic acids can include natural nucleotides (such asA, T/U, C, and G), and can also include analogs of natural nucleotides,such as labeled nucleotides. In one example, a nucleic acid is aninfluenza nucleic acid, which can include nucleic acids purified frominfluenza viruses as well as the amplification products of such nucleicacids.

Nucleotide: The fundamental unit of nucleic acid molecules. A nucleotideincludes a nitrogen-containing base attached to a pentose monosaccharidewith one, two, or three phosphate groups attached by ester linkages tothe saccharide moiety.

The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP orA), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U).

Nucleotides include those nucleotides containing modified bases,modified sugar moieties and modified phosphate backbones, for example asdescribed in U.S. Pat. No. 5,866,336 to Nazarenko et al. (hereinincorporated by reference).

Examples of modified base moieties which can be used to modifynucleotides at any position on its structure include, but are notlimited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N-6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyarninomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine amongstothers.

Examples of modified sugar moieties which may be used to modifynucleotides at any position on its structure include, but are notlimited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or amodified component of the phosphate backbone, such as phosphorothioate,a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or aformacetal or analog thereof.

Primers: Short nucleic acid molecules, such as a DNA oligonucleotide,for example sequences of at least 15 nucleotides, which can be annealedto a complementary target nucleic acid molecule by nucleic acidhybridization to form a hybrid between the primer and the target nucleicacid strand. A primer can be extended along the target nucleic acidmolecule by a polymerase enzyme. Therefore, primers can be used toamplify a target nucleic acid molecule (such as a portion of aninfluenza nucleic acid), wherein the sequence of the primer is specificfor the target nucleic acid molecule, for example so that the primerwill hybridize to the target nucleic acid molecule under very highstringency hybridization conditions.

The specificity of a primer increases with its length. Thus, forexample, a primer that includes 30 consecutive nucleotides will annealto a target sequence with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, to obtain greater specificity,probes and primers can be selected that include at least 15, 20, 25, 30,35, 40, 45, 50 or more consecutive nucleotides.

In particular examples, a primer is at least 15 nucleotides in length,such as at least 15 contiguous nucleotides complementary to a targetnucleic acid molecule. Particular lengths of primers that can be used topractice the methods of the present disclosure (for example, to amplifya region of an influenza nucleic acid) include primers having at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29, at least 30, at least 31, atleast 32, at least 33, at least 34, at least 35, at least 36, at least37, at least 38, at least 39, at least 40, at least 45, at least 50, ormore contiguous nucleotides complementary to the target nucleic acidmolecule to be amplified, such as a primer of 15-60 nucleotides, 15-50nucleotides, or 15-30 nucleotides.

Primer pairs can be used for amplification of a nucleic acid sequence,for example, by PCR, real-time PCR, or other nucleic-acid amplificationmethods known in the art. An “upstream” or “forward” primer is a primer5′ to a reference point on a nucleic acid sequence. A “downstream” or“reverse” primer is a primer 3′ to a reference point on a nucleic acidsequence. In general, at least one forward and one reverse primer areincluded in an amplification reaction. PCR primer pairs can be derivedfrom a known sequence (such as the influenza nucleic acid sequences setforth as SEQ ID NOS:42-50), for example, by using computer programsintended for that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.).

Methods for preparing and using primers are described in, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.; Ausubel et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences. In oneexample, a primer includes a label.

Probe: A probe comprises an isolated nucleic acid capable of hybridizingto a target nucleic acid (such as an influenza nucleic acid). Adetectable label or reporter molecule can be attached to a probe.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes.

Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Intersciences (1987).

In a particular example, a probe includes at least one fluorophore, suchas an acceptor fluorophore or donor fluorophore. For example, afluorophore can be attached at the 5′- or 3′-end of the probe. Inspecific examples, the fluorophore is attached to the base at the 5′-endof the probe, the base at its 3′-end, the phosphate group at its 5′-endor a modified base, such as a T internal to the probe.

Probes are generally at least 20 nucleotides in length, such as at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 41, at least42, at least 43, at least 44, at least 45, at least 46, at least 47, atleast 48, at least 49, at least 50 at least 51, at least 52, at least53, at least 54, at least 55, at least 56, at least 57, at least 58, atleast 59, at least 60, or more contiguous nucleotides complementary tothe target nucleic acid molecule, such as 20-60 nucleotides, 20-50nucleotides, 20-40 nucleotides, or 20-30 nucleotides.

Polymerizing agent: A compound capable of reacting monomer molecules(such as nucleotides) together in a chemical reaction to form linearchains or a three-dimensional network of polymer chains. A particularexample of a polymerizing agent is polymerase, an enzyme which catalyzesthe 5′ to 3′ elongation of a primer strand complementary to a nucleicacid template. Examples of polymerases that can be used to amplify anucleic acid molecule include, but are not limited to the E. coli DNApolymerase I, specifically the Klenow fragment which has 3′ to 5′exonuclease activity, Taq polymerase, reverse transcriptase (such asHIV-1 RT), E. coli RNA polymerase, and wheat germ RNA polymerase II.

The choice of polymerase is dependent on the nucleic acid to beamplified. If the template is a single-stranded DNA molecule, aDNA-directed DNA or RNA polymerase can be used; if the template is asingle-stranded RNA molecule, then a reverse transcriptase (such as anRNA-directed DNA polymerase) can be used.

Quantitating a nucleic acid molecule: Determining or measuring aquantity (such as a relative quantity) of nucleic acid moleculespresent, such as the number of amplicons or the number of nucleic acidmolecules present in a sample. In particular examples, it is determiningthe relative amount or actual number of nucleic acid molecules presentin a sample.

Quenching of fluorescence: A reduction of fluorescence. For example,quenching of a fluorophore's fluorescence occurs when a quenchermolecule (such as the fluorescence quenchers listed above) is present insufficient proximity to the fluorophore that it reduces the fluorescencesignal (for example, prior to the binding of a probe to an influenzanucleic acid sequence, when the probe contains a fluorophore and aquencher).

Real-time PCR: A method for detecting and measuring products generatedduring each cycle of a PCR, which are proportionate to the amount oftemplate nucleic acid prior to the start of PCR. The informationobtained, such as an amplification curve, can be used to determine thepresence of a target nucleic acid (such as an influenza nucleic acid)and/or quantitate the initial amounts of a target nucleic acid sequence.In some examples, real time PCR is real time reverse transcriptase PCR(rt RT-PCR).

In some examples, the amount of amplified target nucleic acid (such asan influenza nucleic acid) is detected using a labeled probe, such as aprobe labeled with a fluorophore, for example a TAQMAN® probe. In thisexample, the increase in fluorescence emission is measured in real time,during the course of the RT-PCR. This increase in fluorescence emissionis directly related to the increase in target nucleic acid amplification(such as influenza nucleic acid amplification). In some examples, thechange in fluorescence (dRn) is calculated using the equationdRn=Rn⁺−Rn⁻, with Rn⁺ being the fluorescence emission of the product ateach time point and Rn⁻ being the fluorescence emission of the baseline.The dRn values are plotted against cycle number, resulting inamplification plots for each sample as illustrated in FIG. 4. Withreference to FIG. 4, the threshold value (Ct) is the PCR cycle number atwhich the fluorescence emission (dRn) exceeds a chosen threshold, whichis typically 10 times the standard deviation of the baseline (thisthreshold level can, however, be changed if desired).

Sample: A sample, such as a biological sample, is a sample obtained froma plant or animal subject. As used herein, biological samples includeall clinical samples useful for detection influenza infection insubjects, including, but not limited to, cells, tissues, and bodilyfluids, such as: blood; derivatives and fractions of blood, such asserum; extracted galls; biopsied or surgically removed tissue, includingtissues that are, for example, unfixed, frozen, fixed in formalin and/orembedded in paraffin; tears; milk; skin scrapes; surface washings;urine; sputum; cerebrospinal fluid; prostate fluid; pus; bone marrowaspirates; bronchoalveolar levage; tracheal aspirates; sputum;nasopharyngeal aspirates; oropharyngeal aspirates; and saliva. Inparticular embodiments, the biological sample is obtained from an animalsubject, such as in the form of bronchoalveolar levage, trachealaspirates, sputum, nasopharyngeal aspirates, oropharyngeal aspirates,and saliva.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn, and tblastx. Blastn is used tocompare nucleic acid sequences, while blastp is used to compare aminoacid sequences. Additional information can be found at the NCBI website.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1554 nucleotidesis 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(i.e., 15÷20*100=75).

1                  20 Target Sequence: atggtggacccggtgggctt(SEQ ID NO: 1) | || ||| |||| |||| | Identified Sequence:acgggggatccggcgggcct (SEQ ID NO: 2)

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions. Stringent conditions are sequence-dependent and aredifferent under different environmental parameters.

The nucleic acid probes and primers disclosed herein are not limited tothe exact sequences shown, as those skilled in the art will appreciatethat changes can be made to a sequence, and not substantially affect theability of the probe or primer to function as desired. For example,sequences having at least 80%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% sequence identity to any ofSEQ ID NOS: 3-38 are provided herein. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is possible that probes and primer can be used that falloutside these ranges.

Signal: A detectable change or impulse in a physical property thatprovides information. In the context of the disclosed methods, examplesinclude electromagnetic signals such as light, for example light of aparticular quantity or wavelength. In certain examples, the signal isthe disappearance of a physical event, such as quenching of light.

TAQMAN® probes: As illustrated in FIG. 3, a linear oligonucleotide probewith a 5′ reporter fluorophore such as 6-carboxyfluorescein (FAM) and a3′ quencher fluorophore, such as BLACKHOLE QUENCHER™ 1 (BHQ™ 1). In theintact TAQMAN® probe, energy is transferred (via FRET) from theshort-wavelength fluorophore to the long-wavelength fluorophore on theother end, quenching the short-wavelength fluorescence. Afterhybridization, the probe is susceptible to degradation by theendonuclease activity of a processing Taq polymerase. Upon degradation,FRET is interrupted, increasing the fluorescence from theshort-wavelength fluorophore and decreasing fluorescence from thelong-wavelength fluorophore.

Target nucleic acid molecule: A nucleic acid molecule whose detection,quantitation, qualitative detection, or a combination thereof, isintended. The nucleic acid molecule need not be in a purified form.Various other nucleic acid molecules can also be present with the targetnucleic acid molecule. For example, the target nucleic acid molecule canbe a specific nucleic acid molecule (which can include RNA such as viralRNA), the amplification of which is intended. Purification or isolationof the target nucleic acid molecule, if needed, can be conducted bymethods known to those in the art, such as by using a commerciallyavailable purification kit or the like. In one example, a target nucleicmolecule is an influenza nucleic acid sequence.

II. Overview of Several Embodiments

Recent increased circulation of highly pathogenic avian influenza, suchas H5N1, in avian populations together with sporadic human infections ofhighly pathogenic avian influenza has raised serious concerns about thepandemic threat of these viruses. The need exists for methods to rapidlydetect and identify influenza viruses, for example to rapidly diagnoseor determine the pandemic potential of viral samples, such as thoseobtained from a subject infected or believed to be infected with aninfluenza virus.

Disclosed herein are methods for the universal detection of allinfluenza type A and type B viruses as well as for the identification ofthe HA genes of influenza A viruses of human health significanceincluding contemporary human H1 and H3, as well as Asian avian H5,Eurasian H7, North American H7, and Asian H9 viruses. The methods havebeen developed in one embodiment with a unique set of nucleic acidprobes and/or primers that are surprisingly effective at detecting anddiscriminating between influenza type A, and type B and subtypes H1, H3,Asian avian H5, North American avian H7, European avian H7, and Asianavian H9 using a variety of conditions. This ability to rapidly screenand identify a virus from among these diverse groups is a significantpublic health advantage.

As disclosed herein, using sequence alignments of all known influenzaviral sequences available, previously unknown regions of high sequencehomology were discovered amongst the individual influenza viral typesand subtypes. These regions were used to create the consensus sequencesshown in FIGS. 9-17. Using these highly homologous regions as a startingpoint the disclosed probes and primers were designed such that they weresurprisingly effective at recognizing genetically diverse influenzaisolates within distinct viral types and/or subtypes. Because of thepandemic potential of influenza subtype Asian avian H5, two regions ofthe H5 HA gene regions of high sequence homology used to designredundant primers and probes.

Probes and Primers

Probes capable of hybridizing to and detecting the presence of influenzanucleic acids are disclosed. The disclosed probes are between 20 and 40nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28 29,30, 31, 32, 32, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length andare capable of hybridizing to the influenza virus nucleic acid. Inseveral embodiments, a probe is capable of hybridizing under very highstringency conditions to an influenza virus nucleic acid sequence setforth as SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50.

In several embodiments, a probe capable of hybridizing to an influenzanucleic acid contains a nucleic acid sequence that is at least 95%identical, such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical to the nucleotide sequence set forth as SEQID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ IDNO:29, SEQ ID NO:32, SEQ ID NO:35, or SEQ ID NO:38. In severalembodiments, a probe capable of hybridizing to an influenza nucleic acidconsists essentially of a nucleic acid sequence set forth as SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ IDNO:29, SEQ ID NO:32, SEQ ID NO:35, or SEQ ID NO:38.

In several embodiments, the probe is influenza type specific. Aninfluenza type specific probe is capable of hybridizing under stringentconditions (such as high stringency, or very high stringency conditions)to an influenza virus nucleic acid from a specific influenza type, suchas influenza type A or type B. For example, a probe that is typespecific for influenza type A (such as specific for an influenza type AM gene sequence, for example the nucleic acid sequence set forth as SEQID NO:42) is not type specific for influenza type B. Likewise, a probethat is type specific for influenza type B (such as specific for aninfluenza type B NS gene sequence, for example the nucleic acid sequenceset forth as SEQ ID NO:43) is not type specific for influenza type A. Inother words a nucleic acid probe that specifically hybridizes to aninfluenza type A nucleic acid (such as a nucleic acid that is at least aportion of the M gene from influenza type A) does not hybridize to aninfluenza type B nucleic acid; such nucleic acids would be type specificprobes for influenza type A. Conversely, a nucleic acid probe thatspecifically hybridizes to an influenza type B nucleic acid (such as anucleic acid that is at least a portion of the NS gene from influenzatype B) does not hybridize to an influenza type A nucleic acid; suchnucleic acids would be type specific probes for influenza type B. Thus,type specific probes can be used to discriminate the presence ofinfluenza type A from influenza type B, or the converse. In someembodiments, the probe is capable of hybridizing under very highstringency conditions to a nucleic acid from influenza A, for example toan influenza type A nucleic acid from the M gene of influenza type A setforth as SEQ ID NO:42. In some embodiments, the probe is capable ofhybridizing under very high stringency conditions to a nucleic acid frominfluenza B, for example to an influenza type B nucleic acid from the NSgene of influenza type B set forth as SEQ ID NO:43.

In some embodiments, the probe is specific for an influenza type Asequence. In a specific example, a probe specific for an influenza typeA nucleic acid includes a nucleic acid sequence at least 95% identicalto SEQ ID NO:8. In some embodiments, the probe is specific for aninfluenza type B sequence. In a specific example, a probe specific foran influenza type B nucleic acid includes a nucleic acid sequence atleast 95% identical to SEQ ID NO:29.

In several embodiments, the probe is influenza subtype specific. Aninfluenza subtype specific probe is capable of hybridizing understringent conditions (such as high stringency, or very high stringencyconditions) to an influenza virus nucleic acid from a specific influenzasubtype, such as influenza subtype H1, H3, H5, North American H7,European H7, or Asian H9. Subtype specific probes can be used to detectthe presence of and differentiate between the various influenzasubtypes. Such probes are specific for one influenza subtype, forexample specific for an influenza HA sequence that is subtype specific,such as an H1, H3, H5, North American H7, European H7, or Asian H9sequence. In some examples, a probe that is subtype specific forinfluenza subtype H1 is not subtype specific for influenza subtype H3,H5, H7 (North American or European), or Asian H9. In another example, aprobe that is subtype specific for influenza subtype H3 is not subtypespecific for influenza subtype H1, H5, H7 (North American or European),or Asian H9. In another example, a probe that is subtype specific forinfluenza subtype H5 is not subtype specific for influenza subtype H1,H3, H7 (North American or European), or Asian H9. In another example, aprobe that is subtype specific for influenza subtype North American H7is not subtype specific for influenza subtype H1, H3, H5, European H7,or Asian H9. In another example, a probe that is subtype specific forinfluenza subtype European H7 is not subtype specific for influenzasubtype H1, H3, H5, North American H7, or Asian H9. In yet anotherexample, a probe that is subtype specific for influenza subtype Asian H9is not subtype specific for influenza subtype H1, H3, H5, or H7 (NorthAmerican or European). To put it another way a nucleic acid probe thatspecifically hybridizes to an influenza subtype H1 nucleic acid does nothybridize to an influenza subtype H3 or any other subtype nucleic acid,such nucleic acids would be type specific probes for influenza type H1.One of skill in the art would understand that the same trend would holdfor the other subtype specific probes.

In some embodiments, the probe is specific for an influenza subtype H1sequence, such as the nucleic acid sequence set forth as SEQ ID NO:44.In a specific example, a probe specific for an influenza subtype H1nucleic acid includes a nucleic acid sequence at least 95% identical toSEQ ID NO:11. In some embodiments, the probe is specific for aninfluenza subtype H3 sequence, such as the nucleic acid sequence setforth as SEQ ID NO:45. In a specific example, a probe specific for aninfluenza subtype H3 nucleic acid includes a nucleic acid sequence atleast 95% identical to SEQ ID NO:14. In some embodiments, the probe isspecific for an influenza subtype H5 sequence, such as the nucleic acidsequence set forth as SEQ ID NO:46. In a specific example, a probespecific for an influenza subtype H5 nucleic acid includes a nucleicacid sequence at least 95% identical to SEQ ID NO:19. In anotherspecific example, a probe specific for an influenza subtype H5 nucleicacid includes a nucleic acid sequence at least 95% identical to SEQ IDNO:24. In some embodiments, the probe is specific for an influenzasubtype North American H7 sequence, such as the nucleic acid sequenceset forth as SEQ ID NO:48. In a specific example, a probe specific foran influenza subtype North American H7 nucleic acid includes a nucleicacid sequence at least 95% identical to SEQ ID NO:32. In someembodiments, the probe is specific for an influenza subtype European H7sequence, such as the nucleic acid sequence set forth as SEQ ID NO:49.In a specific example, a probe specific for an influenza subtypeEuropean H7 nucleic acid includes a nucleic acid sequence at least 95%identical to SEQ ID NO:32. In some embodiments, the probe is specificfor an influenza subtype Asian H9 sequence, such as the nucleic acidsequence set forth as SEQ ID NO:50. In a specific example, a probespecific for an influenza subtype Asian H9 nucleic acid includes anucleic acid sequence at least 95% identical to SEQ ID NO:38.

In some embodiments, the probe is detectably labeled, either with anisotopic or non-isotopic label, alternatively the target nucleic acid(such as an influenza nucleic acid) is labeled. Non-isotopic labels can,for instance, comprise a fluorescent or luminescent molecule, biotin, anenzyme or enzyme substrate or a chemical. Such labels are preferentiallychosen such that the hybridization of the probe with target nucleic acid(such as an influenza nucleic acid) can be detected. In some examples,the probe is labeled with a fluorophore. Examples of suitablefluorophore labels are given above. In some examples, the fluorophore isa donor fluorophore. In other examples, the fluorophore is an accepterfluorophore, such as a fluorescence quencher. In some examples, theprobe includes both a donor fluorophore and an accepter fluorophore.Appropriate donor/acceptor fluorophore pairs can be selected usingroutine methods. In one example, the donor emission wavelength is onethat can significantly excite the acceptor, thereby generating adetectable emission from the acceptor. In some examples, the probe ismodified at the 3′-end to prevent extension of the probe by apolymerase.

In particular examples, the acceptor fluorophore (such as a fluorescencequencher) is attached to the 3′ end of the probe and the donorfluorophore is attached to a 5′ end of the probe. In another particularexample, the acceptor fluorophore (such as a fluorescence quencher) isattached to a modified nucleotide (such as a T) and the donorfluorophore is attached to a 5′ end of the probe.

Primers capable of hybridizing to and directing the amplification ofinfluenza nucleic acids are disclosed. The primers disclosed herein arebetween 15 to 40 nucleotides in length, such as 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, or even 40 nucleotides in length. In several embodiments, a primeris capable of hybridizing under very high stringency conditions to aninfluenza virus nucleic acid sequence set forth as SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, or SEQ ID NO:50, and directing the amplification ofthe influenza nucleic acid.

In several embodiments, a primer capable of hybridizing to and directingthe amplification of an influenza nucleic acid contains a nucleic acidsequence that is at least 95% identical such as at least 96%, at least97%, at least 98%, at least 99%, or even 100% identical to the nucleicacid sequence set forth as SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO: 18, SEQ IDNO: 22, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:37. Inseveral embodiments, a primer capable of hybridizing to an influenzanucleic acid consists essentially of a nucleic acid sequence set forthas SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO:23,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:37.

In several embodiments, the primer is influenza type specific. Aninfluenza type specific primer is capable of hybridizing under stringentconditions (such as high stringency, or very high stringency conditions)to an influenza virus nucleic acid from a specific influenza type, suchas influenza type A or type B. For example, a primer that is typespecific for influenza type A is not type specific for influenza type B.Likewise, a primer that is type specific for influenza type B is nottype specific for influenza type A. In other words a nucleic acid primerthat specifically hybridizes to an influenza type A nucleic acid (suchas a nucleic acid that is at least a portion of the M gene frominfluenza type A, for example the nucleic acid sequence set forth as SEQID NO:42) does not hybridize to an influenza type B nucleic acid, suchnucleic acids would be type specific primers for influenza type A.Conversely, a nucleic acid primer that specifically hybridizes to aninfluenza type B nucleic acid (such as a nucleic acid that is at least aportion of the NS gene from influenza type B, for example the nucleicacid sequence set forth as SEQ ID NO:43) does not hybridize to aninfluenza type A nucleic acid, such nucleic acids would be type specificprimers for influenza type A. Thus, type specific primers can be used tospecifically amplify a nucleic acid from influenza type A or frominfluenza type B, but not both. In some embodiments, the primer iscapable of hybridizing under very high stringency conditions to anucleic acid from influenza A, for example to an influenza type Anucleic acid from the M gene of influenza type A set forth as SEQ IDNO:42. In some embodiments, the primer is capable of hybridizing undervery high stringency conditions to a nucleic acid from influenza B, forexample to an influenza type B nucleic acid from the NS gene ofinfluenza type B set forth as SEQ ID NO:43.

In some embodiments, the primer is specific for an influenza type Asequence, such as an influenza type A M gene sequence. In a specificexample, a primer specific for an influenza type A nucleic acid includesa nucleic acid sequence at least 95% identical to SEQ ID NO:3 or SEQ IDNO:4. In some embodiments, the primer is specific for an influenza typeB sequence, such as an influenza type B NS gene sequence. In a specificexample, a primer specific for an influenza type B nucleic acid includesa nucleic acid sequence at least 95% identical to SEQ ID NO:26 or SEQ IDNO:28.

In several embodiments, the primer is influenza subtype specific. Aninfluenza subtype specific primer is capable of hybridizing understringent conditions (such as high stringency, or very high stringencyconditions) to an influenza virus nucleic acid from a specific influenzasubtype, such as influenza subtype H1, H3, H5, North American H7,European H7 or Asian H9. Such primers are specific for one influenzasubtype, for example specific for an influenza HA sequence that issubtype specific, such as an H1, H3, H5, North American H7, European H7or Asian H9 HA nucleic acid sequence. Subtype specific primers can beused to amplify sequences specific to the various influenza subtypes. Inone example, a primer that is subtype specific for influenza subtype H1is not subtype specific for influenza subtype H3, H5, H7 (North Americanor European), or Asian H9. A primer that is subtype specific forinfluenza subtype H3 is not subtype specific for influenza subtype H1,H5, H7 (North American or European), or Asian H9. A primer that issubtype specific for influenza subtype H5 is not subtype specific forinfluenza subtype H1, H3, H7 (North American or European), or Asian H9.A primer that is subtype specific for influenza subtype North AmericanH7 is not subtype specific for influenza subtype H1, H3, H5, EuropeanH7, or Asian H9. A primer that is subtype specific for influenza subtypeEuropean H7 is not subtype specific for influenza subtype H1, H3, H5,North American H7, or Asian H9. A primer that is subtype specific forinfluenza subtype Asian H9 is not subtype specific for influenza subtypeH1, H3, H5, or H7 (North American or European). To put it another way anucleic acid primer that specifically hybridizes to an influenza subtypeH1 nucleic acid does not hybridize to an influenza subtype H3 or anyother subtype nucleic acid, such nucleic acids would be type specificprimers for influenza type H1. One of skill in the art would understandthat this trend holds for the other subtype specific primers.

In some embodiments, the primer is specific for an influenza subtype H1sequence, such as the nucleic acid sequence set forth as SEQ ID NO:44.In a specific example, a primer specific for an influenza subtype H1nucleic acid includes a nucleic acid sequence at least 95% identical toSEQ ID NO:9 or SEQ ID NO:10. In some examples, the primer is specificfor an influenza subtype H3 sequence, such as the nucleic acid sequenceset forth as SEQ ID NO:45. In a specific example, a primer specific foran influenza subtype H3 nucleic acid includes a nucleic acid sequence atleast 95% identical to SEQ ID NO:12 or SEQ ID NO:13. In some examples,the primer is specific for an influenza subtype H5 sequence, such as thenucleic acid sequence set forth as SEQ ID NO:46. In a specific example,a primer specific for an influenza subtype H5 nucleic acid includes anucleic acid sequence at least 95% identical to SEQ ID NO:17 or SEQ IDNO:18. In a specific example, a primer specific for an influenza subtypeH5 nucleic acid includes a nucleic acid sequence at least 95% identicalto SEQ ID NO:22 or SEQ ID NO:23. In some examples, the primer isspecific for an influenza subtype North American H7 sequence, such asthe nucleic acid sequence set forth as SEQ ID NO:48. In a specificexample, a primer specific for an influenza subtype North American H7nucleic acid includes a nucleic acid sequence at least 95% identical toSEQ ID NO:30 or SEQ ID NO:31. In some examples, the primer is specificfor an influenza subtype European H7 sequence, such as the nucleic acidsequence set forth as SEQ ID NO:49. In a specific example, a primerspecific for an influenza subtype European H7 nucleic acid includes anucleic acid sequence at least 95% identical to SEQ ID NO:33 or SEQ IDNO:34. In some examples, the primer is specific for an influenza subtypeAsian H9 sequence, such as the nucleic acid sequence set forth as SEQ IDNO:50. In a specific example, a primer specific for an influenza subtypeAsian H9 nucleic acid includes a nucleic acid sequence at least 95%identical to SEQ ID NO:36 or SEQ ID NO:38.

In certain embodiments the primers are a set of primers, such as a pairof primers, capable of hybridizing to and amplifying an influenzanucleic acid. Such a set primers comprises at least one forward primerand a least one reverse primer, where the primers are specific for theamplification of an influenza type or subtype nucleic acid. In someexamples, the set of primers includes a pair of primers that is specificfor the amplification of influenza type A, type B, subtype H1, subtypeH3, subtype H5, subtype North American H7, subtype European H7, orsubtype Asian H9.

In certain examples, the pair of primers is specific for theamplification of an influenza type A nucleic acid and includes a forwardprimer at least 95% identical to SEQ ID NO:3 and a reverse primer atleast 95% identical to SEQ ID NO:4. In other examples, the pair ofprimers is specific for the amplification of an influenza subtype H1 andincludes a forward primer at least 95% identical to SEQ ID NO:9 and areverse primer at least 95% identical to SEQ ID NO:10. In otherexamples, the pair of primers is specific for the amplification of aninfluenza subtype H3 and includes a forward primer at least 95%identical to SEQ ID NO:12 and a reverse primer at least 95% identical toSEQ ID NO:13. In other examples, the pair of primers is specific for theamplification of an influenza subtype H5 and includes a forward primerat least 95% identical to SEQ ID NO:17 and a reverse primer at least 95%identical to SEQ ID NO:18. In other examples, the pair of primers isspecific for the amplification of an influenza subtype H5 and includes aforward primer at least 95% identical to SEQ ID NO:22 and a reverseprimer at least 95% identical to SEQ ID NO:23. In other examples, thepair of primers is specific for the amplification of an influenzasubtype type B and includes a forward primer at least 95% identical toSEQ ID NO:26 and a reverse primer at least 95% identical to SEQ IDNO:28. In other examples, the pair of primers is specific for theamplification of an influenza subtype North American H7 and includes aforward primer at least 95% identical to SEQ ID NO:30 and a reverseprimer at least 95% identical to SEQ ID NO:31. In other examples, thepair of primers is specific for the amplification of an influenzasubtype European H7 and includes a forward primer at least 95% identicalto SEQ ID NO:33 and a reverse primer at least 95% identical to SEQ IDNO:34. In other examples, the pair of primers is specific for theamplification of an influenza subtype Asian H9 and includes a forwardprimer at least 95% identical to 95% identical to SEQ ID NO:36 and areverse primer at least 95% identical to SEQ ID NO:38.

Although exemplary probes and primers are provided in SEQ ID NOS:3-38,one skilled in the art will appreciate that the primer and/or probesequence can be varied slightly by moving the probes a few nucleotidesupstream or downstream from the nucleotide positions that they hybridizeto on the influenza nucleic acid, provided that the probe and or primeris still specific for the influenza sequence, such as specific for thetype or subtype of the influenza sequence, for example specific for SEQID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. For example, one ofskill in the art will appreciate that by analyzing the consensussequences shown in FIGS. 9-17 that variations of the probes and primersdisclosed as SEQ ID NOS:3-38 can by made by “sliding” the probes and/orprimers a few nucleotides 5′ or 3′ from their positions, and that suchvariation will still be specific for the influenza viral type and/orsubtype.

Also provided by the present application are probes and primers thatinclude variations to the nucleotide sequences shown in any of SEQ IDNOS:3-38, as long as such variations permit detection of the influenzanucleic acid, such as an influenza type or subtype. For example, a probeor primer can have at least 95% sequence identity such as at least 96%,at least 97%, at least 98%, at least 99% to a nucleic acid consisting ofthe sequence shown in any of SEQ ID NOS:3-38. In such examples, thenumber of nucleotides does not change, but the nucleic acid sequenceshown in any of SEQ ID NOS:3-38 can vary at a few nucleotides, such aschanges at 1, 2, 3, or 4 nucleotides, for example by changing thenucleotides as shown in the tables presented in FIGS. 9-17.

The present application also provides probes and primers that areslightly longer or shorter than the nucleotide sequences shown in any ofSEQ ID NOS:3-38, as long as such deletions or additions permit detectionof the desired influenza nucleic acid, such as an influenza type orsubtype. For example, a probe can include a few nucleotide deletions oradditions at the 5′- or 3′-end of the probe shown in any of SEQ IDNOS:3-38, such as addition or deletion of 1, 2, 3, or 4 nucleotides fromthe 5′- or 3′-end, or combinations thereof (such as a deletion from oneend and an addition to the other end). In such examples, the number ofnucleotides changes. One of skill in the art will appreciate that theconsensus sequences shown in FIGS. 9-17 (SEQ ID NOS:42-50) providesufficient guidance as to what additions and/or subtractions can bemade, while still maintaining specificity for the influenza viral typeand/or subtype.

Detection and Identification of Influenza

A major application of the influenza virus specific primers and probesdisclosed herein is for the detection, typing and subtyping of influenzaviruses in a sample, such as a biological sample obtained from a subjectthat has or is suspected of having an influenza infection. Thus, thedisclosed methods can be used to diagnose if a subject has an influenzainfection and/or discriminate between the viral type and/or subtype thesubject is infected with.

Methods for the detection of influenza nucleic acids are disclosed, forexample to determine if a subject is infected with an influenza virus.Methods also are provided for determining the type and/or subtype of theinfluenza viral nucleic acid, for example to determine the type and/orsubtype of influenza virus a subject is infected with.

The methods described herein may be used for any purpose for whichdetection of influenza is desirable, including diagnostic and prognosticapplications, such as in laboratory and clinical settings. Appropriatesamples include any conventional environmental or biological samples,including clinical samples obtained from a human or veterinary subject,such as a bird. Suitable samples include all biological samples usefulfor detection of viral infection in subjects, including, but not limitedto, cells, tissues (for example, lung, liver and kidney), bone marrowaspirates, bodily fluids (for example, blood, serum, urine,cerebrospinal fluid, bronchoalveolar levage, tracheal aspirates, sputum,nasopharyngeal aspirates, oropharyngeal aspirates, saliva), eye swabs,cervical swabs, vaginal swabs, rectal swabs, stool, and stoolsuspensions. Particularly suitable samples include samples obtained frombronchoalveolar levage, tracheal aspirates, sputum, nasopharyngealaspirates, oropharyngeal aspirates, or saliva. Standard techniques foracquisition of such samples are available. See for example, Schluger etal., J. Exp. Med. 176:1327-33 (1992); Bigby et al., Am. Rev. Respir.Dis. 133:515-18 (1986); Kovacs et al., NEJM 318:589-93 (1988); andOgnibene et al., Am. Rev. Respir. Dis. 129:929-32 (1984).

Detecting an influenza nucleic acid in a sample involves contacting thesample with at least one of the influenza specific probes disclosedherein that is capable of hybridizing to an influenza virus nucleic acidunder conditions of very high stringency (such as a nucleic acid probecapable of hybridizing under very high stringency conditions to aninfluenza nucleic acid sequence set forth as SEQ ID NOS:42-50, forexample a nucleic acid sequence at least 95% identical to the nucleotidesequence set forth as one of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14,SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35,and SEQ ID NO:38), and detecting hybridization between the influenzavirus nucleic acid and the probe. Detection of hybridization between theprobe influenza nucleic acid indicates the presence of the influenzanucleic acid in the sample.

By using influenza type specific probes, the disclosed methods can beused to detect the presence of influenza types in the sample. Forexample, by contacting the sample with an influenza type A specificprobe, such as a probe capable of hybridizing under very high stringencyconditions to an influenza nucleic acid sequence set forth as SEQ IDNO:42, for example a nucleic acid sequence of at least 95% identical toSEQ ID NO:8, and detecting the hybridization of the influenza type Aspecific probe to the influenza nucleic acid, the presence of influenzatype A is detected. Alternatively, contacting the sample with a probespecific for an influenza type B nucleic acid, such as a probe capableof hybridizing under very high stringency conditions to an influenzanucleic acid sequence set forth as SEQ ID NO:43, for example a nucleicacid sequence of at least 95% identical to SEQ ID NO:29, and detectingthe hybridization between the probe and the influenza nucleic acidindicates influenza type B is present. Thus, these disclosed methods canbe used discriminate between the presence of influenza type A or type Bin a sample.

The influenza subtype specific probes disclosed herein can be used todetect the presence of and discriminate between influenza subtypes in asample. For example, contacting a sample with a probe specific forinfluenza subtype H1, such as a probe capable of hybridizing under veryhigh stringency conditions to an influenza nucleic acid sequence setforth as SEQ ID NO:44, for example a nucleic acid at least 95% identicalto the nucleotide sequence set forth as SEQ ID NO:11, and detecting thehybridization between the probe and the influenza nucleic acid indicatesthat influenza subtype H1 is present. In another example, contacting asample with a probe specific for influenza subtype H3, such as a probecapable of hybridizing under very high stringency conditions to aninfluenza nucleic acid sequence set forth as SEQ ID NO:45, for example anucleic acid at least 95% identical to the nucleotide sequence set forthas SEQ ID NO: 14, and detecting the hybridization between the probe andthe influenza nucleic acid indicates the presence of influenza subtypeH3. In another example, contacting a sample with a probe specific forinfluenza subtype H5, such as a probe capable of hybridizing under veryhigh stringency conditions to an influenza nucleic acid sequence setforth as SEQ ID NO:46, for example a nucleic acid at least 95% identicalto the nucleotide sequence set forth as SEQ ID NO:19, and detecting thehybridization between the probe and the influenza nucleic acid indicatesthe presence of influenza subtype H5. In another example, contacting asample with a probe specific for influenza subtype H5, such as a probecapable of hybridizing under very high stringency conditions to aninfluenza nucleic acid sequence set forth as SEQ ID NO:47, for example anucleic acid at least 95% identical to the nucleotide sequence set forthas SEQ ID NO:24, and detecting the hybridization between the probe andthe influenza nucleic acid indicates the presence of influenza subtypeH5. In another example, contacting a sample with a probe specific forinfluenza subtype North American H7, such as a probe capable ofhybridizing under very high stringency conditions to an influenzanucleic acid sequence set forth as SEQ ID NO:48, for example a nucleicacid at least 95% identical to the nucleotide sequence set forth as SEQID NO:32, and detecting the hybridization between the probe and theinfluenza nucleic acid indicates the presence of influenza subtype NorthAmerican H7. In yet another example, contacting a sample with a probespecific for influenza subtype European H7, such as a probe capable ofhybridizing under very high stringency conditions to an influenzanucleic acid sequence set forth as SEQ ID NO:49, for example a nucleicacid at least 95% identical to the nucleotide sequence set forth as SEQID NO:35, and detecting the hybridization between the probe and theinfluenza nucleic acid indicates the presence of influenza subtypeEuropean H7. In still another example, contacting a sample with a probespecific for influenza subtype Asian H9, such as a probe capable ofhybridizing under very high stringency conditions to an influenzanucleic acid sequence set forth as SEQ ID NO:50, for example a nucleicacid at least 95% identical to the nucleotide sequence set forth as SEQID NO:38, and detecting the hybridization between the probe and theinfluenza nucleic acid indicates the presence of influenza subtype AsianH9.

In some embodiments, detecting the presence of an influenza nucleic acidsequence in a sample includes the extraction of influenza RNA. RNAextraction relates to releasing RNA from a latent or inaccessible formin a virion, cell or sample and allowing the RNA to become freelyavailable. In such a state, it is suitable for effective detectionand/or amplification of the influenza nucleic acid. Releasing RNA mayinclude steps that achieve the disruption of virions containing viralRNA, as well as disruption of cells that may harbor such virions.Extraction of RNA is generally carried out under conditions thateffectively exclude or inhibit any ribonuclease activity that may bepresent. Additionally, extraction of RNA may include steps that achieveat least a partial separation of the RNA dissolved in an aqueous mediumfrom other cellular or viral components, wherein such components may beeither particulate or dissolved.

One of ordinary skill in the art will know suitable methods forextracting RNA from a sample; such methods will depend upon, forexample, the type of sample in which the influenza RNA is found. Forexample, the RNA may be extracted using guanidinium isothiocyanate, suchas the single-step isolation by acid guanidiniumisothiocyanate-phenol-chloroform extraction of Chomczynski et al. (Anal.Biochem. 162:156-59, 1987). The sample can be used directly or can beprocessed, such as by adding solvents, preservatives, buffers, or othercompounds or substances. Viral RNA can be extracted using standardmethods. For instance, rapid RNA preparation can be performed using acommercially available kit (such as the Roche MagNA Pure Compact NucleicAcid Isolation Kit I, QIAAMP® Viral RNA Mini Kit, QIAAMP® MinElute VirusSpin Kit or RNEASY® Mini Kit (QIAGEN); NUCLISENS® NASBA Diagnostics(bioMérieux); MASTERPURE™ Complete DNA and RNA Purification Kit(EPICENTRE). Alternatively, an influenza virion may be disrupted by asuitable detergent in the presence of proteases and/or inhibitors ofribonuclease activity. Additional exemplary methods for extracting RNAare found, for example, in World Health Organization, Manual for thevirological investigation of polio, World Health Organization, Geneva,2001.

In some embodiments, the probe is detectably labeled, either with anisotopic or non-isotopic label; in alternative embodiments, theinfluenza nucleic acid is labeled. Non-isotopic labels can, forinstance, comprise a fluorescent or luminescent molecule, or an enzyme,co-factor, enzyme substrate, or hapten. The probe is incubated with asingle-stranded or double-stranded preparation of RNA, DNA, or a mixtureof both, and hybridization determined. In some examples thehybridization results in a detectable change in signal such as inincrease or decrease in signal, for example from the labeled probe.Thus, detecting hybridization comprises detecting a change in signalfrom the labeled probe during or after hybridization relative to signalfrom the label before hybridization.

In some embodiments, influenza nucleic acids present in a sample areamplified prior to using a hybridization probe for detection. Forinstance, it can be advantageous to amplify a portion of the influenzanucleic acid, then detect the presence of the amplified influenzanucleic acid. For example, to increase the number of nucleic acids thatcan be detected, thereby increasing the signal obtained. Influenzaspecific nucleic acid primers can be used to amplify a region that is atleast about 50, at least about 60, at least about 70, at least about 80at least about 90, at least about 100, at least about 200, or more basepairs in length to produce amplified influenza specific nucleic acids.Any nucleic acid amplification method can be used to detect the presenceof influenza in a sample. In one specific, non-limiting example,polymerase chain reaction (PCR) is used to amplify the influenza nucleicacid sequences. In other specific, non-limiting examples, real-time PCR,reverse transcriptase-polymerase chain reaction (RT-PCR), real-timereverse transcriptase-polymerase chain reaction (rt RT-PCR), ligasechain reaction, or transcription-mediated amplification (TMA) is used toamplify the influenza nucleic acid. In a specific example, the influenzavirus nucleic acid is amplified by rt RT-PCR. Techniques for nucleicacid amplification are well-known to those of skill in the art.

Typically, at least two primers are utilized in the amplificationreaction, however it is envisioned that one primer can be utilized, forexample to reverse transcribe a single stranded nucleic acid such as asingle-stranded influenza RNA. Amplification of the influenza nucleicacid involves contacting the influenza nucleic acid with one or moreprimers that are capable of hybridizing to and directing theamplification of an influenza nucleic acid (such as a nucleic acidcapable of hybridizing under very high stringency conditions to aninfluenza nucleic acid set forth as SEQ NO:42-50, for example a primerthat is least 95% identical to the nucleotide sequence set forth as oneof SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24,SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38). In someembodiments, the sample is contacted with at least one primer that isspecific for an influenza type or subtype, such as those disclosedherein.

In some embodiments, the sample is contacted with at least one pair ofprimers that include a forward and reverse primer that both hybridize toan influenza nucleic acid specific for an influenza viral type and orsubtype, such as influenza type A, type B, subtype H3, H5, H7 (NorthAmerican or European), or Asian H9. Examples of suitable primer pairsfor the amplification of influenza type and/or subtype specific nucleicacids are described above.

Any type of thermal cycler apparatus can be used for the amplificationof the influenza nucleic acids and/or the determination ofhybridization. Examples of suitable apparatuses include a PTC-100®Peltier Thermal Cycler (MJ Research, Inc.; San Francisco, Calif.), aROBOCYCLER® 40 Temperature Cycler (Stratagene; La Jolla, Calif.), or aGENEAMP® PCR System 9700 (Applied Biosystems; Foster City, Calif.). Forreal-time PCR, any type of real-time thermocycler apparatus can be used.For example, a BioRad iCycler iQ™, LIGHTCYCLER™ (Roche; Mannheim,Germany), a 7700 Sequence Detector (Perkin Elmer/Applied Biosystems;Foster City, Calif.), ABI™ systems such as the 7000, 7500, 7700, or 7900systems (Applied Biosystems; Foster City, Calif.), or an MX4000™,MX3000™ or MX3005™ (Stratagene; La Jolla, Calif.), and CepheidSMARTCYCLER™ can by used to amplify nucleic acid sequences in real-time.

The amplified influenza nucleic acid, for example an influenza type orsubtype specific nucleic acid, can be detected in real-time, for exampleby real-time PCR such as real-time RT-PCR, in order to determine thepresence, the identity, and/or the amount of an influenza type orsubtype specific nucleic acid in a sample. In this manner, an amplifiednucleic acid sequence, such as an amplified influenza nucleic acidsequence, can be detected using a probe specific for the productamplified from the influenza sequence of interest, such as an influenzasequence that is specific for influenza type A, type B, subtype H1, H3,H5, North America H7, European H7, and Asian H9. Detecting the amplifiedproduct includes the use of labeled probes that are sufficientlycomplementary and hybridize to the amplified nucleic acid sequence.Thus, the presence, amount, and/or identity of the amplified product canbe detected by hybridizing a labeled probe, such as a fluorescentlylabeled probe, complementary to the amplified product. In oneembodiment, the detection of a target nucleic acid sequence of interestincludes the combined use of PCR amplification and a labeled probe suchthat the product is measured using real-time RT-PCR. In anotherembodiment, the detection of an amplified target nucleic acid sequenceof interest includes the transfer of the amplified target nucleic acidto a solid support, such as a blot, for example a Northern blot, andprobing the blot with a probe, for example a labeled probe, that iscomplementary to the amplified target nucleic acid sequence. In yetanother embodiment, the detection of an amplified target nucleic acidsequence of interest includes the hybridization of a labeled amplifiedtarget nucleic acid to probes disclosed herein that are an arrayed in apredetermined array with an addressable location and that arecomplementary to the amplified target nucleic acid.

In one embodiment, the fluorescently-labeled probes rely uponfluorescence resonance energy transfer (FRET), or in a change in thefluorescence emission wavelength of a sample, as a method to detecthybridization of a DNA probe to the amplified target nucleic acid inreal-time. For example, FRET that occurs between fluorogenic labels ondifferent probes (for example, using HybProbes) or between a fluorophoreand a non-fluorescent quencher on the same probe (for example, using amolecular beacon or a TAQMAN® probe) can identify a probe thatspecifically hybridizes to the DNA sequence of interest and in this way,using Influenza type and/or subtype specific probes, can detect thepresence, identity, and/or amount of an influenza type and/or subtype ina sample. In one embodiment, the fluorescently-labeled DNA probes usedto identify amplification products have spectrally distinct emissionwavelengths, thus allowing them to be distinguished within the samereaction tube.

In another embodiment, a melting curve analysis of the amplified targetnucleic acid can be performed subsequent to the amplification process.The T_(m) of a nucleic acid sequence depends on the length of thesequence and its G/C content. Thus, the identification of the T_(m) fora nucleic acid sequence can be used to identify the amplified nucleicacid.

Influenza Profiling Arrays

An array containing a plurality of heterogeneous probes for thedetection, typing, and/or subtyping of influenza viruses are disclosed.Such arrays may be used to rapidly detect and/or identify the typeand/or subtype of an influenza virus in a sample. For example the arrayscan be used to determine the presence of influenza A or influenza B in asample and to determine if the influenza virus is of subtype H1, H3, H5,H7 (North American or European), or Asian H9.

Arrays are arrangements of addressable locations on a substrate, witheach address containing a nucleic acid, such as a probe. In someembodiments, each address corresponds to a single type or class ofnucleic acid, such as a single probe, though a particular nucleic acidmay be redundantly contained at multiple addresses. A “microarray” is aminiaturized array requiring microscopic examination for detection ofhybridization. Larger “macroarrays” allow each address to berecognizable by the naked human eye and, in some embodiments, ahybridization signal is detectable without additional magnification. Theaddresses may be labeled, keyed to a separate guide, or otherwiseidentified by location.

In some embodiments, an influenza profiling array is a collection ofseparate probes at the array addresses. The influenza profiling array isthen contacted with a sample suspected of containing influenza nucleicacids under conditions allowing hybridization between the probe andnucleic acids in the sample to occur. Any sample potentially containing,or even suspected of containing, influenza nucleic acids may be used,including nucleic acid extracts, such as amplified or non-amplified DNAor RNA preparations. A hybridization signal from an individual addresson the array indicates that the probe hybridizes to a nucleotide withinthe sample. This system permits the simultaneous analysis of a sample byplural probes and yields information identifying the influenza nucleicacids contained within the sample. In alternative embodiments, the arraycontains influenza nucleic acids and the array is contacted with asample containing a probe. In any such embodiment, either the probe orthe influenza nucleic acids may be labeled to facilitate detection ofhybridization.

The nucleic acids may be added to an array substrate in dry or liquidform. Other compounds or substances may be added to the array as well,such as buffers, stabilizers, reagents for detecting hybridizationsignal, emulsifying agents, or preservatives.

In certain examples, the array includes one or more molecules or samplesoccurring on the array a plurality of times (twice or more) to providean added feature to the array, such as redundant activity or to provideinternal controls.

Within an array, each arrayed nucleic acid is addressable, such that itslocation may be reliably and consistently determined within the at leastthe two dimensions of the array surface. Thus, ordered arrays allowassignment of the location of each nucleic acid at the time it is placedwithin the array. Usually, an array map or key is provided to correlateeach address with the appropriate nucleic acid. Ordered arrays are oftenarranged in a symmetrical grid pattern, but nucleic acids could bearranged in other patterns (for example, in radially distributed lines,a “spokes and wheel” pattern, or ordered clusters). Addressable arrayscan be computer readable; a computer can be programmed to correlate aparticular address on the array with information about the sample atthat position, such as hybridization or binding data, including signalintensity. In some exemplary computer readable formats, the individualsamples or molecules in the array are arranged regularly (for example,in a Cartesian grid pattern), which can be correlated to addressinformation by a computer.

An address within the array may be of any suitable shape and size. Insome embodiments, the nucleic acids are suspended in a liquid medium andcontained within square or rectangular wells on the array substrate.However, the nucleic acids may be contained in regions that areessentially triangular, oval, circular, or irregular. The overall shapeof the array itself also may vary, though in some embodiments it issubstantially flat and rectangular or square in shape.

Influenza profiling arrays may vary in structure, composition, andintended functionality, and may be based on either a macroarray or amicroarray format, or a combination thereof. Such arrays can include,for example, at least 10, at least 25, at least 50, at least 100, ormore addresses, usually with a single type of nucleic acid at eachaddress. In the case of macroarrays, sophisticated equipment is usuallynot required to detect a hybridization signal on the array, thoughquantification may be assisted by standard scanning and/orquantification techniques and equipment. Thus, macroarray analysis asdescribed herein can be carried out in most hospitals, agricultural andmedial research laboratories, universities, or other institutionswithout the need for investment in specialized and expensive readingequipment.

Examples of substrates for the arrays disclosed herein include glass(e.g., functionalized glass), Si, Ge, GaAs, GaP, SiO₂, SiN₄, modifiedsilicon nitrocellulose, polyvinylidene fluoride, polystyrene,polytetrafluoroethylene, polycarbonate, nylon, fiber, or combinationsthereof. Array substrates can be stiff and relatively inflexible (forexample glass or a supported membrane) or flexible (such as a polymermembrane). One commercially available product line suitable for probearrays described herein is the Microlite line of MICROTITER® platesavailable from Dynex Technologies UK (Middlesex, United Kingdom), suchas the Microlite 1+96-well plate, or the 384 Microlite+ 384-well plate.

Addresses on the array should be discrete, in that hybridization signalsfrom individual addresses can be distinguished from signals ofneighboring addresses, either by the naked eye (macroarrays) or byscanning or reading by a piece of equipment or with the assistance of amicroscope (microarrays).

Addresses in an array may be of a relatively large size, such as largeenough to permit detection of a hybridization signal without theassistance of a microscope or other equipment. Thus, addresses may be assmall as about 0.1 mm across, with a separation of about the samedistance. Alternatively, addresses may be about 0.5, 1, 2, 3, 5, 7, or10 mm across, with a separation of a similar or different distance.Larger addresses (larger than 10 mm across) are employed in certainembodiments. The overall size of the array is generally correlated withsize of the addresses (for example, larger addresses will usually befound on larger arrays, while smaller addresses may be found on smallerarrays). Such a correlation is not necessary, however.

The arrays herein may be described by their densities (the number ofaddresses in a certain specified surface area). For macroarrays, arraydensity may be about one address per square decimeter (or one address ina 10 cm by 10 cm region of the array substrate) to about 50 addressesper square centimeter (50 targets within a 1 cm by 1 cm region of thesubstrate). For microarrays, array density will usually be one or moreaddresses per square centimeter, for instance, about 50, about 100,about 200, about 300, about 400, about 500, about 1000, about 1500,about 2,500, or more addresses per square centimeter.

The use of the term “array” includes the arrays found in DNA microchiptechnology. As one, non-limiting example, the probes could be containedon a DNA microchip similar to the GENECHIP® products and relatedproducts commercially available from Affymetrix, Inc. (Santa Clara,Calif.). Briefly, a DNA microchip is a miniaturized, high-density arrayof probes on a glass wafer substrate. Particular probes are selected,and photolithographic masks are designed for use in a process based onsolid-phase chemical synthesis and photolithographic fabricationtechniques similar to those used in the semiconductor industry. Themasks are used to isolate chip exposure sites, and probes are chemicallysynthesized at these sites, with each probe in an identified locationwithin the array. After fabrication, the array is ready forhybridization. The probe or the nucleic acid within the sample may belabeled, such as with a fluorescent label and, after hybridization, thehybridization signals may be detected and analyzed.

Kits

The nucleic acid primers and probes disclosed herein can be supplied inthe form of a kit for use in the detection, typing, and/or subtyping ofinfluenza, including kits for any of the arrays described above. In sucha kit, an appropriate amount of one or more of the nucleic acid probesand or primers is provided in one or more containers or held on asubstrate. A nucleic acid probe and/or primer may be provided suspendedin an aqueous solution or as a freeze-dried or lyophilized powder, forinstance. The container(s) in which the nucleic acid(s) are supplied canbe any conventional container that is capable of holding the suppliedform, for instance, microfuge tubes, ampoules, or bottles. The kits caninclude either labeled or unlabeled nucleic acid probes for use indetection, typing, and subtyping of influenza nucleotide sequences.

In some applications, one or more primers (as described above), such aspairs of primers, may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers. Withsuch an arrangement, the sample to be tested for the presence ofinfluenza nucleic acids can be added to the individual tubes andamplification carried out directly.

The amount of nucleic acid primer supplied in the kit can be anyappropriate amount, and may depend on the target market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each nucleic acid primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. General guidelines for determining appropriate amounts may befound in Innis et al., Sambrook et al., and Ausubel et al. A kit mayinclude more than two primers in order to facilitate the PCRamplification of a larger number of influenza nucleotide sequences.

In some embodiments, kits also may include the reagents necessary tocarry out PCR amplification reactions, including DNA sample preparationreagents, appropriate buffers (such as polymerase buffer), salts (forexample, magnesium chloride), and deoxyribonucleotides (dNTPs).

One or more control sequences for use in the PCR reactions also may besupplied in the kit (for example, for the detection of human RNAse P).

Particular embodiments include a kit for detecting and typing and/orsubtyping an influenza nucleic acid based on the arrays described above.Such a kit includes at least one probe specific for an influenza nucleicacid (as described above) and instructions. A kit may contain more thanone different probe, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 25, 50, 100, or more probes. The instructions may includedirections for obtaining a sample, processing the sample, preparing theprobes, and/or contacting each probe with an aliquot of the sample. Incertain embodiments, the kit includes an apparatus for separating thedifferent probes, such as individual containers (for example,microtubules) or an array substrate (such as, a 96-well or 384-wellmicrotiter plate). In particular embodiments, the kit includesprepackaged probes, such as probes suspended in suitable medium inindividual containers (for example, individually sealed EPPENDORF®tubes) or the wells of an array substrate (for example, a 96-wellmicrotiter plate sealed with a protective plastic film). In otherparticular embodiments, the kit includes equipment, reagents, andinstructions for extracting and/or purifying nucleotides from a sample.

Synthesis of Oligonucleotide Primers and Probes

In vitro methods for the synthesis of oligonucleotides are well known tothose of ordinary skill in the art; such methods can be used to produceprimers and probes for the disclosed methods. The most common method forin vitro oligonucleotide synthesis is the phosphoramidite method,formulated by Letsinger and further developed by Caruthers (Caruthers etal., Chemical synthesis of deoxyoligonucleotides, in Methods Enzymol.154:287-313, 1987). This is a non-aqueous, solid phase reaction carriedout in a stepwise manner, wherein a single nucleotide (or modifiednucleotide) is added to a growing oligonucleotide. The individualnucleotides are added in the form of reactive 3′-phosphoramiditederivatives. See also, Gait (Ed.), Oligonucleotide Synthesis. Apractical approach, IRL Press, 1984.

In general, the synthesis reactions proceed as follows: Adimethoxytrityl or equivalent protecting group at the 5′ end of thegrowing oligonucleotide chain is removed by acid treatment. (The growingchain is anchored by its 3′ end to a solid support such as a siliconbead.) The newly liberated 5′ end of the oligonucleotide chain iscoupled to the 3′-phosphoramidite derivative of the next deoxynucleotideto be added to the chain, using the coupling agent tetrazole. Thecoupling reaction usually proceeds at an efficiency of approximately99%; any remaining unreacted 5′ ends are capped by acetylation so as toblock extension in subsequent couplings. Finally, the phosphite triestergroup produced by the coupling step is oxidized to the phosphotriester,yielding a chain that has been lengthened by one nucleotide residue.This process is repeated, adding one residue per cycle. See, forexample, U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679, and5,132,418. Oligonucleotide synthesizers that employ this or similarmethods are available commercially (for example, the PolyPlexoligonucleotide synthesizer from Gene Machines, San Carlos, Calif.). Inaddition, many companies will perform such synthesis (for example,Sigma-Genosys, The Woodlands, Tex.; Qiagen Operon, Alameda, Calif.;Integrated DNA Technologies, Coralville, Iowa; and TriLinkBioTechnologies, San Diego, Calif.).

The following examples are provided to illustrate particular features ofcertain embodiments. However, the particular features described belowshould not be construed as limitations on the scope of the invention,but rather as examples from which equivalents will be recognized bythose of ordinary skill in the art.

EXAMPLES Example 1 Sample Collection and Preparation

This example describes exemplary procedures for the collection andpreparation of samples for the determination of the presence ofinfluenza nucleic acids.

Samples obtained from the respiratory tract were collected either asbroncheoalveolar lavage, tracheal aspirates, sputum, nasopharyngeal ororopharyngeal aspirates or washes, or nasopharyngeal or oropharyngealswabs. Swabs were collected using swabs with a DACRON® tip and analuminum or plastic shaft. For specific viral isolates, viruses werepropagated in either MDCK cells or embryonated chicken eggs. Forvalidation of the primers and probes disclosed herein the followingviral isolates were used: X31 (H3N2)(Aichi/2/68×PR8 reassortant),A/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (H1N1),A/Vietnam/1203/2003 (H5N1), A/HongKong/1203/99 (H9N2),A/Netherlands/219/2003 (H7N7), A/New York/5295/2003 (H7N2) and B/HongKong/330/2001. Samples were refrigerated or frozen prior to nucleic acidextraction. Viral RNA was extracted from the samples using the QIAAMP®Viral RNAEASY™ Mini Kit available from QIAGEN® (Valencia, Calif.)according to the manufacturer's recommendations.

Example 2 Selection of Probe/Primer Sets

This example describes the rational and procedures used to design probesand primers for the detection, typing and subtyping of influenza virus.

Oligonucleotide primers and probes for universal detection of influenzatype A and influenza type B influenza viruses were selected from highlyconserved (consensus) regions of the M and NS genes, respectively, basedon nucleotide alignments of all available sequence data from GENBANK®database of National Centers for Biological Information, NIH (NCBI) andthe Influenza Sequence Database of Los Alamos National Laboratories(LANL). Similarly, primers and probes specific for the hemagglutinin(HA) gene of modern human H1, H3, Asian avian H5, North American avianH7, European avian H7 and Asian avian H9 viruses were designed. Becauseof the pandemic potential of influenza subtype Asian avian H5, tworedundant primer and probe set were designed to detect this influenzasubtype. The consensus sequence for the region of the influenza type A Mgene used for the design of probes and primers specific for influenzatype A is given in the table shown in FIGS. 9A-9F. The consensussequence for the region of the influenza type B NS gene used for thedesign of probes and primers specific for influenza type B is given inthe table shown in FIGS. 10A-10I. The consensus sequences for the regionof the influenza subtype H1 HA gene used for the design of probes andprimers specific for influenza subtype H1 is given in the table shown inFIGS. 11A-11F. The consensus sequences for the region of the influenzasubtype H3 HA gene used for the design of probes and primers specificfor influenza subtype H3 is given in the table shown in FIGS. 12A-12F.The consensus sequence for a region of the influenza subtype H5 HA geneused for the design of probes and primers specific for influenza tsubtype H5 is given in the table shown in FIGS. 13A-13I. The consensussequence for a region of the influenza subtype H5 HA gene used for thedesign of probes and primers specific for influenza subtype H5 is givenin the table shown in FIGS. 14A-14L. The consensus sequence for theregion of the influenza subtype North American H7 HA gene used for thedesign of probes and primers specific for influenza subtype NorthAmerican H7 is given in the table shown in FIGS. 15A-15B. The consensussequence for the region of the influenza subtype European H7 HA geneused for the design of probes and primers specific for influenza subtypeEuropean H7 is given in the table shown in FIGS. 16A-16C. The consensussequence for the region of the influenza subtype Asian H9 HA gene usedfor the design of probes and primers specific for influenza subtypeAsian H9 is given in the table shown in FIGS. 17A-171. With reference toFIGS. 9-17 the consensus sequence for the influenza viral type orsubtype specific nucleic acid is shown at the top of each table. Theboxed sequences represent the positions of exemplary probes and primersdisclosed herein. Nucleotide variations for the indicated influenzaviral isolates are shown in the columns below the consensus sequence,with a dot meaning that the nucleotide present at that position isidentical to the consensus sequence. In addition K=G or T; S=G or C; R=Aor G; M=A or C; and Y=T or C.

In order to avoid loss of reaction performance due to primer-dimer orhairpin loop formation, primers and probes were evaluated using Softwarepackages PRIMEREXPRESS® (Applied Biosystems) and BEACON DESIGNER 4.0®(PREMIER Biosoft International) to predict secondary structures andself-annealing probabilities.

Each primer and probe sequence was subjected to a nucleotide Blastsearch (NCBI) against the entire GENBANK® nucleotide database tovalidate their specificities and avoid non-specific reactivity. Theprobe and primers listed in Table 1 were selected for validation usingTAQMAN® chemistry. Primers and dual-labeled TAQMAN® probes (Table 1)were synthesized by the Biotechnology Core Facility, Centers for DiseaseControl.

TABLE 1 Probe and Primer Sets Sequence SEQ ID NO Flu AFlu A Forward Primer GAC CRA TCC TGT CAC CTC TGA C  3Flu A Consensus Reverse AGG X₁CA TTY TGG ACA AAK CGT  4 Primer CTA X₂X₃Flu A Reverse Primer No. 1 AGG GCA TTY TGG ACA AAK CGT  5 CTAFlu A Reverse Primer No. 2 AGG CAT TYT GGA CAA AKC GTC  6 TAC GFlu A Reverse Primer No. 3 GGG CAT TYT GGA CAA AKC GTC  7 TAC GFlu A Probe¹ TGC AGT CCT CGC TCA CTG GGC ACG  8 H1 H1 Forward PrimerAAC TAC TAC TGG ACT CTR CTK GAA  9 H1 Reverse PrimerCCA TTG GTG CAT TTG AGK TGA TG 10 H1 Probe²TGA YCC AAA GCC ″T″CT ACT CAG 11 TGC GAA AGC H3 H3 Forward PrimerAAG CAT TCC YAA TGA CAA ACC 12 H3 Reverse PrimerATT GCR CCR AAT ATG CCT CTA GT 13 H3 Consensus Probe¹CAG SAT CAC ATA TGG GSC CTG TCC 14 CAG H3 Probe No. 1¹CAG GAT CAC ATA TGG GSC CTG TCC 15 CAG H3 Probe No. 2¹CAG CAT CAC ATA TGG GSC CTG TCC 16 CAG H5 primer and probe set aH5 a Consensus Forward TGG AAA GTR TAA RAA ACG GAA 17 Primer CGTH5 a Consensus Reverse YGC TAG GGA RCT CGC CAC TG 18 PrimerH5 a Consensus Probe² YRA CTA YCC GCA G″T″A TTC AGA 19AGA AGC AAG AYT AA H5 a Probe1² TGA CTA CCC GCA G″T″A TTC AGA 20AGA AGC AAG ACT AA H5 a Probe2² CAA CTA TCC GCA G″T″A TTC AGA 21AGA AGC AAG ATT AA H5 primer and probe set b H5 b Consensus ForwardGGA ATG YCC CAA ATA YGT GAA 22 Primer RTC AA H5 b Consensus ReverseCTC CCC TGC TCR TTG CTA TGG T 23 Primer H5 b Consensus Probe²TAY CCA TAC CAA CCA ″T″CT ACC 24 ATT CCC TGC CAT H5 b Probe No. 1²TAC CCA TAC CAA CCA ″T″CT ACC 25 ATT CCC TGC CAT Flu BFlu B Consensus Forward TCC TCA AYT CAC TCT TCG AGC G 26 PrimerFlu B Forward Primer No. 1 TCC TCA ACT CAC TCT TCG AGC G 27Flu B Reverse Primer CGG TGC TCT TGA CCA AAT TGG 28 Flu B Probe¹CCA ATT CGA GCA GCT GAA ACT 29 GCG GTG H7 North America H7 ForwardAAA TGC ACA AGG AGA GGG AAC TG 30 Primer North America H7 ReverseCAT TGC YAC YAA SAG YTC AGC RT 31 Primer North America H7 Probe²AAA GCA CCC ART C″T″G CAA TAG 32 ATC AGA TCA CAG GC European H7 ForwardGCT TCA GGC ATC AAA ATG CAC 33 Primer AAG G European H7 Reverse PrimerCAT TGC TAC YAA GAG TTC AGC RT 34 European H7 Probe²ACC ACA CTT CTG TCA ″T″GG AAT 35 CTC TGG TCC A H9Asian H9 Forward Primer CAA GCT GGA ATC TGA RGG AAC 36 TTA CAAsian H9 Reverse Primer GCA TCT GCA AGA TCC ATT GGA CAT 37Asian H9 Probe¹ CCC AGA ACA RGA AGG CAG CAA 38 ACC CCA TTG RNPRNP Forward Primer AGA TTT GGA CCT GCG AGC G 39 RNP Reverse PrimerGAG CGG CTG TCT CCA CAA GT 40 RNP Probe¹ TTC TGA CCT GAA GGC TCT GCG CG41

Where K=G or T; S=G or C; R=A or G; Y=T or C; X₁=G or no nucleotide;X₂=C or no nucleotide; and X3=G or no nucleotide. ¹TAQMAN® probes werelabeled at the 5′-end with the reporter molecule 6-carboxyfluorescein(FAM) and with the quencher, BLACKHOLE QUENCHER™ 1 (BHQ™1) (BiosearchTechnologies, Inc., Novato, Calif.) at the 3′-end. ²TAQMAN® probes werelabeled at the 5′-end with the reporter molecule 6-carboxyfluorescein(FAM) and quenched internally at a modified “T” residue with QSY® 7(Molecular Probes, Inc.) or BHQ™. Internally quenched probes also weremodified at the 3′-end to prevent extension of the probe by Taqpolymerase.

The reaction efficiency of the primer sets was individually tested in aset of five-fold serial dilutions of viral RNA using SYBER green bindingto double stranded nucleic acids as an indicator of amplification. Allrt RT-PCR assays for detection and characterization of influenza weredesigned to achieve reaction efficiencies of approximately 100%. Areaction efficiency of 100% indicates that a primer set is capable ofachieving a complete doubling of the nucleic acid target sequence in asingle round of amplification.

With reference to FIG. 5A-5C, the reaction efficiency of the primer setfor universal detection of influenza type A was determined by testingagainst a five-fold serial dilution of viral RNA. Identical tests werecarried out with the primer sets specific for each viral type andsubtype. FIG. 5 A shows the relative fluorescence of SYBER green whenbound to double stranded nucleic acid versus the number of PCR cycles.The individual rt RT-PCR reactions were subjected to melting curveanalysis to confirm that the SYBER green fluorescence was attributableto specific amplification of the influenza A gene target. As shown inFIG. 5B, all reactions showed double stranded nucleic acid melting atthe same temperature, indicating specific amplification. Similar meltingcurve analysis was performed for all primer sets and demonstrated thatthe primers were specific for their specific target influenza nucleicacid sequence. As shown on FIG. 5C, reaction Ct values for the influenzaA specific primers were plotted against their relative RNA concentrationand the doubling efficiency (% reaction efficiency) was determined byestimating the slope using regression analysis. A slope of 3.23indicates a reaction efficiency of approximately 100%. A reactionefficiency of 100.3% percent was obtained for the influenza type Aspecific primers. All primer sets tested had a reaction efficiency ofapproximately 100% when subjected to the same analysis.

Following the validation of the reaction specificity and efficiency ofthe primer sets the reaction efficiency of the primer/probe sets wasvalidated. Using a five-fold viral dilutions series the reactionefficiency of the individual influenza type and subtype primer/probesets was analyzed. Exemplary data for the analysis of the probe/primerset specific for influenza type A is shown in FIG. 6A and FIG. 6B.

As shown in FIG. 6A, the reaction efficiency of the primer/probe set foruniversal detection of type A influenza was determined by testingagainst a five-fold serial dilution of viral RNA. The reaction Ct valueswere plotted against their relative RNA concentration to estimate thereaction efficiency using regression analysis (FIG. 6B). Similar testwere carried out on all available primer sets. As shown in Table 2, allof the primer/probe sets exhibited reaction efficiencies at or near100%.

TABLE 2 rt RT-PCT reaction efficiencies. Efficiency R Squared InfluenzaTyping sets Flu A 100.3% 1.000 Flu B 100.7% 1.000 Influenza Subtypingsets Human H1 HA 100.1% 0.996 Human H3 HA 99.8% 0.998 Eurasian H5 HA (a)102.8% 0.998 Eurasian H5 HA (b) 100.3% 0.996 North American Avian H794.2% 1.000 HA Eurasian H7 HA 98.9% 0.994 Asian Avian H9 HA 96.4% 0.995

One of the design criteria for the disclosed primer and probe sets wasthat they could be used at a variety of annealing (hybridization)temperatures. Thus, the probe/primer sets were tested for their abilityto perform at a range of annealing temperatures from 50-62.5° C. FIG. 7shows a plot of the real-time RT-PCR reactivity comparison of theinfluenza A primer/probe set with annealing temperatures ranging from50-62.5° C. In order to determine the optimal thermocycling conditions,each probe/primer set was similarly tested with annealing temperaturesranging from 50-62.5° C. All primer/probe sets were designed todemonstrate comparable reactivity at annealing temperatures ranging from50-60° C. and exhibited stable Ct values at all temperatures tested(Table 3 and Table 4).

TABLE 3 Thermal gradient analysis of TAQMAN ® primer/probe sets from50-62.5 C. Flu A Flu B H1 H3 AsH5a Tm Ct ΔCt Ct ΔCt Ct ΔCt Ct ΔCt Ct ΔCt50 21.6 0.2 21.6 −0.1 27.1 0.1 25.6 0.0 19.3 −0.1 50 21.4 0 21.9 0.227.6 0.6 25.6 0.0 19.3 −0.1 51 21.3 −0.1 21.8 0.1 26.6 −0.4 25.6 0.019.3 −0.1 51 21.4 0 21.3 −0.4 27.1 0.1 25.6 0.0 19.2 −0.2 52.5 21.2 −0.221.7 0.0 26.4 −0.6 25.5 −0.1 19.3 −0.1 52.5 21.5 0.1 21.5 −0.2 27.1 0.125.5 −0.1 19.2 −0.2 54.8 21.2 −0.2 21.8 0.1 27 0.0 25.6 0.0 19.2 −0.254.8 21.3 −0.1 21.5 −0.2 26.9 −0.1 25.5 −0.1 19.3 −0.1 58 21.4 0 21.90.2 27 0.0 25.7 0.1 19.6 0.2 58 21.5 0.1 21.4 −0.3 26.9 −0.1 25.6 0.019.4 0.0 60.3 21.4 0 22.2 0.5 27.1 0.1 25.7 0.1 19.8 0.4 60.3 21.6 0.222 0.3 27.5 0.5 25.7 0.1 19.8 0.4 61.7 21.4 0 22.5 0.8 27.4 0.4 26.3 0.720.3 0.9 61.7 21.7 0.3 21.9 0.2 27.9 0.9 26.2 0.6 20.3 0.9 62.5 21.6 0.222.3 0.6 28.2 1.2 26.4 0.8 20.4 1.0 62.5 21.5 0.1 22.6 0.9 27.4 0.4 26.71.1 20.6 1.2 Mean Ct Mean Ct Mean Ct Mean Ct Mean Ct 21.4 21.7 27.0 25.619.4 NAmH7 EurH7 AsH9 AsH5b Ct ΔCt Ct ΔCt Ct ΔCt Ct ΔCt 50 27.3 0.3 25.70.1 27.4 −0.1 20 0.6 50 27.4 0.4 26.2 0.6 27.6 0.1 20.2 0.8 51 27 0.025.5 −0.1 27.7 0.2 19.7 0.3 51 26.9 −0.1 25.9 0.3 27.5 0.0 19.8 0.4 52.526.8 −0.2 25.4 −0.2 27.6 0.1 19.6 0.2 52.5 26.9 −0.1 25.7 0.1 27.6 0.119.4 0.0 54.8 26.5 −0.5 25.2 −0.4 27.6 0.1 19.1 −0.3 54.8 26.9 −0.1 25.4−0.2 27.3 −0.2 19.2 −0.2 58 26.8 −0.2 25.8 0.2 27.6 0.1 18.9 −0.5 5827.3 0.3 25.1 −0.5 27.3 −0.2 19 −0.4 60.3 27 0.0 26.1 0.5 27.2 −0.3 18.8−0.6 60.3 27.4 0.4 24.7 −0.9 27.4 −0.1 18.9 −0.5 61.7 27.6 0.6 26.4 0.827.2 −0.3 19 −0.4 61.7 27.6 0.6 24.5 −1.1 27.1 −0.4 18.9 −0.5 62.5 21.10.7 25.6 0.0 27.1 −0.4 18.7 −0.7 62.5 27.4 0.4 24.2 −1.4 27.3 −0.2 19−0.4 Mean Ct Mean Ct Mean Ct Mean Ct 27.0 25.6 27.5 19.4 Mean Ct = meanof Ct value from reactions with Tm 50-60.0 C.; ΔCt = Ct value − Mean Ct

TABLE 4 Thermal gradient analysis of TAQMAN ® primer/probe combinedstatistics. Flu A Flu B H1 H3 AsH5a NAmH7 EurH7 AsH9 AsH5b Tm AvΔCtAvΔCt AvΔCt AvΔCt AvΔCt AvΔCt AvΔCt AvΔCt AvΔCt 50 0.10 0.03 0.32 0.00−0.14 0.33 0.39 0.02 0.72 51 −0.05 −0.07 −0.18 0.00 −0.14 −0.07 0.140.12 0.37 52.5 −0.05 −0.12 −0.28 −0.10 −0.14 −0.17 −0.01 0.12 0.12 54.8−0.15 −0.07 −0.08 −0.05 −0.14 −0.32 −0.26 −0.03 −0.23 58 0.05 −0.07−0.08 0.05 0.11 0.03 −0.11 −0.03 −0.43 60.3 0.10 0.38 0.27 0.10 0.410.18 −0.16 −0.18 −0.53 61.7 0.15 0.48 0.62 0.65 0.91 0.58 −0.11 −0.33−0.43 62.5 0.15 0.73 0.77 0.95 1.11 0.53 −0.66 −0.28 −0.53

Example 3 Real-Time Reverse Transcriptase (Rt RT-PCR) of Samples

This example describes the procedures used for the determination of thepresence of influenza types and subtypes in a sample using rt RT-PCR ina multiwell format.

Hydrolysis probe (TAQMAN®) rt RT-PCR reactions were performed usingQUANTITECT™ Probe One-step RT-PCR (QIAGEN®) and TAQMAN® One-Step RT-PCRMaster Mix (ABI) kits according to manufacturer's recommendedprocedures. Primer and probe reaction concentrations were 0.8 μM and 0.2μM, respectively.

Individual 1.5 ml microcentrifuge tubes were prepared for eachindividual primer/probe set used. Individual primers and probes werevortexed and briefly centrifuged prior to dispensing. Into eachmicrocentrifuge tube was added 20 microliter rt RT-PCR master mix,wherein the master mix was optimized for various real time PCRinstruments. The mister mix was prepared as shown in Table 5.

TABLE 5 Master Mix. Invitrogen/Bi ABI Qiagen orad 2X PCR Master Mix N ×12.5 μl N × 12.5 μl N × 12.5 μl RT Mix N × 0.625 μl N × 0.25 μl N × 0.5μl Forward primer (0.8 μM final N × 0.5 μl N × 0.5 μl N × 0.5 μlconcentration) Reverse primer (0.8 μM final N × 0.5 μl N × 0.5 μl N ×0.5 μl concentration) Probe (0.2 μM final N × 0.5 μl N × 0.5 μl N × 0.5μl concentration) Nuclease free water N × 5.375 μl N × 5.75 μl N × 5.5μl Total volume N × 20.0 μl N × 20.0 μl N × 20.0 μlWhere N is the number of samples including non template controls (NTC).For viral template controls (VTC) and positive controls for human RNAseP individual mastermixes were prepared. The reactions were mixed bypipeting up and down, without vortexing. Twenty microliters of eachmaster mix was added into individual wells of a 96 well plate. Anexample of the arrayed format used is shown in Table 6 below:

TABLE 6 Probe/Primer Setup for rt RT-PCR. 1 2 3 4 5 6 7 8 9 10 11 12 AFluA FluA FluA FluA FluA FluA FluA FluA FluA FluA FluA FluA B H1 H1 H1H1 H1 H1 H1 H1 H1 H1 H1 H1 C H3 H3 H3 H3 H3 H3 H3 H3 H3 H3 H3 H3 D H5aH5a H5a H5a H5a H5a H5a H5a H5a H5a H5a H5a E H5b H5b H5b H5b H5b H5bH5b H5b H5b H5b H5b H5b F H9 H9 H9 H9 H9 H9 H9 H9 H9 H9 H9 H9 G FluBFluB FluB FluB FluB FluB FluB FluB FluB FluB FluB FluB H RNP RNP RNP RNPRNP RNP RNP RNP RNP RNP RNP RNPFluA is a primer probe set specific for influenza A; H1 is a primerprobe set specific for H1; H3 is a primer probe set specific for H3; H5ais a primer probe set specific for H5; H5b is a primer probe setspecific for H5; H9 is a primer probe set specific for H9; FluB is aprimer probe set specific for influenza B; and RNP is a primer probe setspecific for human RNAse P.

RNA Samples of viral unknown as well as the NTC, VTC, and a mockextraction control were added to the individual wells. NTCs were addedfirst to control for contamination in the master mix. For NTCs 5microliters of distilled water was added. Five microliters of viralunknown was added to each well with the exception of control wells. Forpositive controls five microliters of viral RNA was added. The mockextraction controls were added after the samples have been added tocontrol for cross-contamination during sample preparation or addition.VTCs were added last after all samples and NTCs were sealed to preventcontamination. An example of the array format used is shown in Table 7below:

TABLE 7 Sample Setup. 1 2 3 4 5 6 7 8 9 10 11 12 A NTC S1 S2 S3 S4 S5 S6S7 S8 S9 MOCK VTC B NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 MOCK VTC C NTC S1 S2S3 S4 S5 S6 S7 S8 S9 MOCK VTC D NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 MOCK VTCE NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 MOCK VTC F NTC S1 S2 S3 S4 S5 S6 S7 S8S9 MOCK VTC G NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 MOCK VTC H NTC S1 S2 S3 S4S5 S6 S7 S8 S9 MOCK VTCWhere NTC is the non-template control (no RNA). S1-S9 are samplesobtained from a subject(s). MOCK is a mock extraction control and VTC isthe viral template control.

Example 4 Real Time RT-PCR of Samples

This example describes rt-RT-PCR parameters used for the determinationof the presence, type and subtype of influenza in a sample.

Prior to an rt RT-PCR run, the 96 well plate was centrifuged at 500×gfor 30 seconds at 4° C. The plate was loaded into a thermocycler andsubjected to the PCR cycle as shown in Table 8. All reactions wereperformed on a Stratagene MX4000™, MX3000P™ or BioRad IQ ICYCLER™platform. PCR conditions were optimized for each of the listedinstruments. The reaction volume was 25 μl.

TABLE 8 Optimized PCR Conditions. ABI Invitrogen/Biorad Qiagen Reverse50° C. for 30 min 60° C. for 5 min 60° C. for 5 min Transcription 50° C.for 30 min 50° C. for 30 min Taq inhibitor 95° C. for 10 min 95° C. for2 min 95° C. for 15 min inactivation PCR 95° C. for 15 sec 95° C. for 15sec 95° C. for 15 sec amplification (45 55° C. for 30 sec* 55° C. for 30sec* 55° C. for 30 sec* cycles) 72° C. for 30 sec 72° C. for 30 sec 72°C. for 30 sec *Fluorescence data was collected during the 55° C.incubation step. Primer/Probe sets performed comparably at annealingtemperatures ranging from 50-60° C.

Example 5 The Determination of Influenza Viral Type and Subtype inSamples Obtained from Subjects

This example describes the determination of the presence, type, andsubtype of influenza viral nucleic acid in samples obtained fromsubjects.

Samples obtained from four subjects were tested for the presence ofinfluenza using influenza specific probe and primer sets disclosedherein in rt RT-PCR TAQMAN® assays. In addition, the samples were testedfor the presence of influenza viral types A and B and influenza subtypesH1, H2, and H5. The detection of human RNAse P was used as a control.

The rt RT-PCR data obtained for samples 1, 2, and 3 is shown in FIGS.8A, 8B, and 8C respectively. FIG. 8A shows the rt RT-PCR runs for sample1, which was determined to be positive for influenza type A subtype H5.FIG. 8B shows the rt RT-PCR runs for sample 2, which was determined tobe positive for influenza type A subtype H3. FIG. 8C shows the rt RT-PCRruns for sample 3s, which was determined to not contain influenza.

The tabulated results are shown in Table 9 below.

TABLE 9 Influenza type and subtype in samples obtained from subjects.Flu A H1 H3 H5 Flu B RNP Results Sample 1 + − − + − + A/H5 Sample 2 +− + − − + A/H3 Sample 3 − − − − − + Not detected Sample 4 − − − − − −Invalid

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used, andit is intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds,chemical moieties, or examples described in conjunction with aparticular aspect, embodiment, or example of the invention are to beunderstood to be applicable to any other aspect, embodiment, or exampleof the invention. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims.

We claim:
 1. A probe and primer set, comprising: a probe consisting ofthe nucleotide sequence set forth as SEQ ID NO: 14, 15, or 16 and atleast one attached label, wherein the at least one attached labelcomprises a radioactive isotope, enzyme substrate, co-factor, ligand,chemiluminescent agent, fluorophore, fluorescence quencher, hapten,enzyme, chemical, or combination thereof; a primer consisting of thenucleic acid sequence shown in SEQ ID NO: 12; and a primer consisting ofthe nucleic acid sequence shown in SEQ ID NO:
 13. 2. The probe andprimer set of claim 1, wherein the at least one attached label comprisesa fluorophore.
 3. The probe and primer set of claim 1, wherein the atleast one attached label comprises a fluorophore and a fluorescencequencher.
 4. The probe and primer set of claim 1, wherein thefluorophore is 6-carboxyfluorescein.
 5. The probe and primer set ofclaim 1, wherein the at least one attached label comprises biotin. 6.The probe and primer set of claim 3, wherein the probe comprises thefluorophore on its 5′-end and the fluorescence quencher on its 3′-end.7. A kit for detecting an influenza virus nucleic acid molecule in asample, comprising: a probe consisting of the nucleotide sequence setforth as SEQ ID NO: 14, 15, or 16 and at least one attached label,wherein the at least one attached label comprises a radioactive isotope,enzyme substrate, co-factor, ligand, chemiluminescent agent,fluorophore, fluorescence quencher, hapten, enzyme, chemical, orcombination thereof; a primer consisting of the nucleic acid sequenceshown in SEQ ID NO: 12; and a primer consisting of the nucleic acidsequence shown in SEQ ID NO:
 13. 8. The kit of claim 7, wherein the kitcomprises an array that comprises the probe, or a device comprising anarray that comprises the probe.
 9. The kit of claim 7, wherein the kitfurther comprises a human RNAse P control probe.
 10. The kit of claim 7,wherein the at least one attached label comprises a fluorophore.
 11. Thekit of claim 7, wherein the at least one attached label comprises afluorophore and a fluorescence quencher.
 12. The kit of claim 11,wherein the probe comprises the fluorophore on its 5′-end and thefluorescence quencher on its 3′-end.
 13. A method for diagnosing aninfluenza virus infection in a subject suspected of having an influenzainfection, comprising: contacting a sample comprising nucleic acidmolecules obtained from the subject with the probe and primer set ofclaim 1; amplifying influenza virus nucleic acid molecules in the samplewith the primers, thereby generating amplified influenza virus nucleicacid molecules; detecting hybridization between the amplified influenzavirus nucleic acid molecules and the probe; and determining that thesubject is infected with influenza virus when hybridization between theamplified influenza virus nucleic acid molecules and the probe isdetected.
 14. The method of claim 13, further comprising: discriminatingbetween an influenza type A infection and an influenza type B infection;and/or discriminating between an influenza subtype H1 infection, aninfluenza subtype H3 infection, an influenza subtype North American H7infection, an influenza subtype European H7 infection, and an influenzasubtype Asian H9 infection.
 15. The method of claim 13, whereindetecting hybridization of the probe to the influenza virus nucleic acidsequence set forth as SEQ ID NO: 45 indicates the presence of influenzaH3 in the sample.
 16. The method of claim 13, wherein detectinghybridization comprises detecting a change in signal from the labeledprobe during or after hybridization relative to signal from the labelbefore hybridization.
 17. The method of claim 13, wherein the amplifyingcomprises polymerase chain reaction (PCR), real-time PCR, reversetranscriptase-polymerase chain reaction (RT-PCR), real-time reversetranscriptase-polymerase chain reaction (rt RT-PCR), ligase chainreaction, or transcription-mediated amplification (TMA).
 18. The methodof claim 13, wherein the sample is obtained from bronchoalveolar lavage,tracheal aspirates, sputum, nasopharyngeal aspirates, oropharyngealaspirates, or saliva.
 19. The method of claim 13, wherein the probe isarrayed in a predetermined array with an addressable location.
 20. Themethod of claim 13, wherein the at least one attached label comprises afluorophore.
 21. The method of claim 13, wherein the at least oneattached label comprises a fluorophore and a fluorescence quencher. 22.A method for detecting influenza virus nucleic acid molecules in asample, comprising: contacting the sample with the probe and primer setof claim 1; amplifying influenza virus nucleic acid molecules in thesample with the primers, thereby generating amplified influenza virusnucleic acid molecules; detecting hybridization between the amplifiedinfluenza virus nucleic acid molecules and the probe; and determiningthat the influenza virus nucleic acid molecules are present in thesample when hybridization between the amplified influenza virus nucleicacid molecules and the probe is detected.
 23. The method of claim 22,wherein detecting hybridization of the probe to the influenza virusnucleic acid sequence set forth as SEQ ID NO: 45 indicates the presenceof influenza H3 in the sample.
 24. The method of claim 22, whereindetecting hybridization comprises detecting a change in signal from thelabeled probe during or after hybridization relative to signal from thelabel before hybridization.
 25. The method of claim 22, wherein theamplifying comprises polymerase chain reaction (PCR), real-time PCR,reverse transcriptase-polymerase chain reaction (RT-PCR), real-timereverse transcriptase-polymerase chain reaction (rt RT-PCR), ligasechain reaction, or transcription-mediated amplification (TMA).
 26. Themethod of claim 22, wherein the sample is a biological sample obtainedfrom a subject.
 27. The method of claim 26, wherein the presence of aninfluenza virus nucleic acid in the biological sample indicates thepresence of an influenza virus infection in the biological sampleobtained from the subject.
 28. The method according to claim 26, whereinthe biological sample is obtained from bronchoalveolar lavage, trachealaspirates, sputum, nasopharyngeal aspirates, oropharyngeal aspirates, orsaliva.
 29. The method of claim 22, wherein the probe is arrayed in apredetermined array with an addressable location.
 30. The method ofclaim 22, wherein the at least one attached label comprises afluorophore.
 31. The method of claim 22, wherein the at least oneattached label comprises a fluorophore and a fluorescence quencher.